Lpxc inhibitor and methods of making

ABSTRACT

Provided herein is an LpxC inhibitor compound, as well as methods of making and pharmaceutical compositions comprising said compound, and methods of use thereof in the treatment of disease that would benefit from treatment with an LpxC inhibitor, including gram-negative bacterial infections such as urinary tract infections and the like.

CROSS-REFERENCE

This application is a continuation of Ser. No. 17/211,029, filed Mar.24, 2021, which claims the benefit of U.S. Provisional PatentApplication No. 62/994,654, filed Mar. 25, 2020, and U.S. ProvisionalPatent Application No. 63/153,152, filed Feb. 24, 2021, each of whichare incorporated herein by reference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under AI120246 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

A need exists in the medicinal arts for the effective treatment ofillness caused by bacterial infection.

BRIEF SUMMARY OF THE INVENTION

Provided herein is an LpxC inhibitor compound, as well as methods ofmaking and pharmaceutical compositions comprising said compound, andmethods of use thereof in the treatment of disease that would benefitfrom treatment with an LpxC inhibitor, including gram-negative bacterialinfections such as urinary tract infections and the like.

In one aspect, disclosed herein is a process for the preparation ofFormula 15:

-   -   wherein X is halogen, —OTf, —OTs, or —OMs; and    -   each PG is a suitable protecting group;

comprising:

-   -   (1) contacting the compound of Formula 14:

-   -   -   wherein X is halogen, —OTf, —OTs, or —OMs; and        -   each PG is a suitable protecting group;

    -   with a suitable oxidation reagent system in a suitable solvent        to provide a compound of Formula 15.

In some embodiments, the suitable oxidation reagent system of step (1)is a TEMPO/bleach system; and the suitable solvent of step (1) isacetonitrile, dichloromethane, chloroform, dichloroethane, hexanes,water, or a combination thereof. In some embodiments, the suitableoxidation reagent system of step (1) comprises catalytic TEMPO andstoichiometric bleach; and the suitable solvent of step (1) is a mixtureof water and dichloromethane. In some embodiments, step (1) is performedat a temperature of from about 0° C. to about 5° C.

In some embodiments, the compound of Formula 14 is prepared by:

-   -   (1a) contacting a compound of Formula 13.

-   -   -   wherein X is halogen, —OTf, —OTs, or —OMs;        -   R₁ is C₁-C₁₀ alkyl, aryl, or benzyl; and        -   each PG is a suitable protecting group;

    -   with a suitable borohydride reagent in a suitable solvent to        provide the compound of Formula 14.

In some embodiments, the suitable borohydride reagent of step (1a) islithium borohydride, sodium borohydride, sodium cyanoborohydride,potassium borohydride, lithium triethylborohydride, or sodiumtriacetoxyborohydride; and the suitable solvent of step (1a) isacetonitrile, methanol, ethanol, isopropyl alcohol, dimethoxyethane,2-methyltetrahydrofuran, methyl tert-butyl ether, cyclopentyl methylether, tetrahydrofuran, diethyl ether, diisopropyl ether, 1,4-dioxane,toluene, water, or a combination thereof. In some embodiments, thesuitable borohydride reagent of step (1a) is lithium borohydride; andthe suitable solvent of step (1a) is a mixture of tetrahydrofuran andethanol. In some embodiments, the step (1a) is performed at atemperature of from about 0° C. to about 25° C.

In some embodiments, the process further comprises crystallizing thecompound of Formula 14. In some embodiments, the compound of Formula 14is crystallized from acetonitrile, methanol, ethanol, isopropyl alcohol,acetone, methyl acetate, ethyl acetate, dichloromethane, chloroform,diethyl ether, diisopropyl ether, tert-butyl methyl ether,tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, benzene, toluene,petroleum ether, pentane, hexane, heptane, cyclohexane, acetic acid,water, or a mixture thereof. In some embodiments, the compound ofFormula 14 is crystallized from a mixture of isopropyl alcohol andwater.

In some embodiments, the compound of Formula 13 is prepared by:

-   -   (1b) contacting a compound of Formula 12:

-   -   -   wherein X is halogen, —OTf, —OTs, or —OMs; and        -   R₁ is C₁-C₁₀ alkyl, aryl, or benzyl;

    -   with a compound of Formula 9:

-   -   -   wherein LG is a suitable leaving group; and        -   each PG is a suitable protecting group;

    -   in the presence of a suitable base, and in a suitable solvent,        to provide a compound of Formula 13.

In some embodiments, the suitable base of step (1b) is n-butyl lithium,lithium diisopropylamide (LDA), lithium bis(trimethylsilyl)amide(LiHMDS), or lithium tetramethylpiperidide (LiTMP); and the suitablesolvent of step (1b) is diethyl ether, diisopropyl ether,tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, tert-butyl methylether, cyclopropyl methyl ether, or a combination thereof. In someembodiments, the suitable base of step (1b) is LiHDMS; and the suitablesolvent of step (1b) is tetrahydrofuran. In some embodiments, step (1b)is performed at a temperature of about −25 to about −15° C. In someembodiments, in step (1b), the compound of Formula 12 is reacted withthe base for from about 15 min to about 60 min before the addition ofthe compound of Formula 9.

In some embodiments, the process further comprises (2) contacting thecompound of Formula 15 with a compound of Formula 16, or a salt thereof:

-   -   in the presence of a suitable reducing agent in a suitable        solvent to provide a compound of Formula 17:

-   -   wherein X is halogen, —OTf, —OTs, or —OMs; and    -   each PG is a suitable protecting group.

In some embodiments, the suitable reducing agent of step (2) is sodiumborohydride, sodium cyanoborohydride, sodium triacetoxyborohydride,lithium cyanoborohydride, H₂/catalyst, or picoline-borane; and thesuitable solvent of step (2) is acetonitrile, methanol, ethanol,dichloromethane, chloroform, dichloroethane, toluene, water, or acombination thereof. In some embodiments, the suitable reducing agent ofstep (2) is picoline-borane; and the suitable solvent of step (2) is amixture of methanol and dichloromethane. In some embodiments, step (2)is performed at a temperature of from about 0° C. to about 25° C.

In some embodiments, the process further comprises (3) contacting thecompound of Formula 17 with a compound of Formula 20, or a salt thereof:

-   -   in the presence of a coupling catalyst, a suitable base, and in        a suitable solvent to provide a compound of Formula 21:

-   -   wherein each PG is a suitable protecting group.

In some embodiments, the coupling catalyst of step (3) is a palladiumcatalyst; the suitable base of step (3) is sec-butylamine ortetrabutylammonium fluoride (TBAF); and the suitable solvent of step (3)is acetonitrile, dimethylformamide, diethyl ether, ethanol,tetrahydrofuran, isopropyl alcohol, 1,4-dioxane, toluene, water, or acombination thereof. In some embodiments, the coupling catalyst of step(3) is a palladium catalyst; the suitable base of step (3) issec-butylamine; and the suitable solvent of step (3) is water. In someembodiments, step (3) is performed at a temperature of about 40-45° C.

In some embodiments, the process further comprises (4) contacting thecompound of Formula 21 with a suitable reagent in a suitable solvent toprovide(S)-1-(3-(5,6-dihydroxypyrimidin-4-yl)-2-(4-((4-(morpholinomethyl)phenyl)ethynyl)phenyl)propyl)azetidine-3-carbonitrile(Compound A):

In some embodiments, the suitable reagent of step (4) is H₂/catalyst,HCl, HBr, TFA, TBAF, BCl₃, 9-1-BBN, BF₃—OEt₂, TMS-Cl, or TMS-Br; and thesuitable solvent of step (4) is acetonitrile, dichloromethane,chloroform, dichloroethane, diethyl ether, tetrahydrofuran, isopropylalcohol, 1,4-dioxane, toluene, anisole, water, or a combination thereof.In some embodiments, the suitable reagent of step (4) is TFA; and thesuitable solvent of step (4) is anisole. In some embodiments, step (4)further comprises pentamethylbenzene.

In some embodiments, the process further comprises crystallizingCompound A. In some embodiments, Compound A is crystallized fromacetonitrile, methanol, ethanol, isopropyl alcohol, acetone, methylacetate, ethyl acetate, dichloromethane, chloroform, diethyl ether,diisopropyl ether, tert-butyl methyl ether, tetrahydrofuran,2-methyltetrahydrofuran, dioxane, benzene, toluene, petroleum ether,pentane, hexane, heptane, cyclohexane, acetic acid, water, or a mixturethereof. In some embodiments, Compound A is crystallized from a mixtureof ethyl acetate and tetrahydrofuran.

In some embodiments, the process further comprises treatment of thecompound of Formula 21 with a metal scavenger. In some embodiments, theprocess further comprises treatment of Compound A with a metalscavenger. In some embodiments, the metal scavenger comprises SiO₂,charcoal, aqueous solution of L-cysteine, a Silicycle metal scavenger,Si-thiol, SiliaBond DMT, SiliaBond Cysteine, or 3-mercaptopropyl ethylsulfide silica.

In some embodiments, LG is a halogen, a sulfonate, or a sulfate. In someembodiments, LG is Cl, Br, I, —OTf, —OTs, or —OMs.

In some embodiments, each —O-PG is a benzyl ether, an acetal, or a silylether; wherein each PG is the same protecting group. In someembodiments, each PG is benzyl, p-methoxybenzyl, methoxymethyl,[2-(trimethylsilyl)ethoxy]methyl, triisopropylsilyl, ortert-butyldimethylsilyl.

In some embodiments, X is Cl, Br, I, —OTf, —OTs, or —OMs. In someembodiments, X is Cl, Br, or I.

In some embodiments, R₁ is methyl, isopropyl, tert-butyl, phenyl, orbenzyl.

In some embodiments, LG is Br; each PG is Bn; X is I; and R₁ is benzyl.

In another aspect, disclosed herein is a process for the preparation ofa compound of Formula 4:

-   -   wherein PG is a suitable protecting group; and    -   R is C₁-C₁₀ alkyl;    -   comprising:    -   (1) contacting the compound of Formula 3:

-   -   -   wherein PG is a suitable protecting group; and        -   R is C₁-C₁₀ alkyl;

    -   with formamidine acetate and a suitable base in a suitable        solvent to provide the compound of Formula 4.

In some embodiments, the suitable base of step (1) is sodium hydride,sodium methoxide, sodium ethoxide, lithium methoxide, lithium ethoxide,n-butyl lithium, lithium diisopropylamide (LDA), lithiumbis(trimethylsilyl)amide (LiHMDS), or lithium tetramethylpiperidide(LiTMP); and the suitable solvent of step (1b) is methanol, ethanol,isopropyl alcohol, diethyl ether, diisopropyl ether, tetrahydrofuran,2-methyltetrahydrofuran, 1,4-dioxane, tert-butyl methyl ether,cyclopropyl methyl ether, or a combination thereof. In some embodiments,the suitable base of step (1) is sodium ethoxide; and the suitablesolvent of step (1) is ethanol. In some embodiments, step (1) isperformed at a temperature of about 0° C. to 10° C.

In some embodiments, the process further comprises (2) contacting thecompound of Formula 4 with a halogenating agent and a suitable base in asuitable solvent to provide the compound of Formula 5:

-   -   wherein PG is a suitable protecting group; and    -   R is C₁-C₁₀ alkyl; and    -   X′ is Cl or Br.

In some embodiments, the halogenating agent of step (2) is POCl₃, POBr₃,or SOCl₂; the suitable base of step (2) is triethylamine,diisopropylethylamine, sec-butylamine, 1,2,2,6,6-pentamethylpiperidine,tributylamine, or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); and thesuitable solvent of step (2) is acetonitrile, dichloromethane,chloroform, dichloroethane, toluene, or a combination thereof. In someembodiments, the suitable halogenating agent of step (2) is POCl₃; thesuitable base of step (2) is triethylamine; and the suitable solvent ofstep (2) is toluene. In some embodiments, step (1) is performed at atemperature of about 85° C. to 95° C.

In some embodiments, the process further comprises (3) contacting thecompound of Formula 5 with benzyl alcohol in the presence of a suitablebase, and in a suitable solvent to provide a compound of Formula 6-I orFormula 7-I, or a combination thereof:

-   -   wherein PG is a suitable protecting group; and    -   R is C₁-C₁₀ alkyl.

In some embodiments, the suitable base of step (3) is triethylamine,diisopropylethylamine, sec-butylamine, 1,2,2,6,6-pentamethylpiperidine,tributylamine, or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); and thesuitable solvent of step (3) is acetonitrile, dimethylformamide, diethylether, ethanol, tetrahydrofuran, isopropyl alcohol, 1,4-dioxane,toluene, water, or a combination thereof. In some embodiments, thesuitable base of step (3) is DBU; and the suitable solvent of step (3)is acetonitrile. In some embodiments, step (3) is performed at atemperature of about 20° C. to 25° C.

In some embodiments, the process further comprises (4) contacting thecompound of Formula 6-I or Formula 7-I, or combination thereof, with asuitable reducing agent in a suitable solvent to provide a compound ofFormula 8-I:

-   -   wherein PG is a suitable protecting group.

In some embodiments, the reducing agent of step (4) is sodiumborohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, orlithium cyanoborohydride; and the suitable solvent of step (4) isacetonitrile, dimethylformamide, diethyl ether, methanol, ethanol,tetrahydrofuran, isopropyl alcohol, 1,4-dioxane, toluene, water, or acombination thereof. In some embodiments, the reducing agent of step (4)is sodium borohydride; and the suitable solvent of step (4) is a mixtureof isopropyl alcohol and methanol. In some embodiments, step (4) isperformed at a temperature of about 0° C. to room temperature.

In some embodiments, the process further comprises (5) contacting thecompound of Formula 8-1 with a suitable reagent in a suitable solvent toprovide the compound of Formula 9-I:

-   -   wherein PG is a suitable protecting group; and    -   LG is a suitable leaving group.

In some embodiments, the suitable reagent of step (5) is a halogenatingagent, a sulfonating agent, or a sulfonyl chloride. In some embodiments,the suitable reagent of step (5) is a halogenating agent; and LG is ahalogen. In some embodiments, the suitable reagent of step (5) is SOCl₂,PBr₃, or PCl₃; and the suitable solvent is acetonitrile,dimethylformamide, diethyl ether, ethanol, tetrahydrofuran, isopropylalcohol, 1,4-dioxane, toluene, water, or a combination thereof. In someembodiments, the suitable reagent of step (5) is PBr₃; and the suitablesolvent is dimethylformamide. In some embodiments, step (5) furthercomprises a suitable base, selected from pyridine, N-methylmorpholine,triethylamine, diisopropylethylamine, sec-butylamine,1,2,2,6,6-pentamethylpiperidine, tributylamine, and1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). In some embodiments, thesuitable base is pyridine.

In some embodiments, R is methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, t-butyl, pentyl, iso-amyl, hexyl, heptyl, octyl,or nonyl. In some embodiments, R is methyl or ethyl.

In some embodiments, —O-PG is a benzyl ether, an acetal, or a silylether. In some embodiments, PG is benzyl, p-methoxybenzyl,methoxymethyl, [2-(trimethylsilyl)ethoxy]methyl, triisopropylsilyl, ortert-butyldimethylsilyl. In some embodiments, PG is benzyl.

In some embodiments, R is ethyl; PG is benzyl; and X′ is Cl.

Other objects, features and advantages of the compounds, methods andcompositions described herein will become apparent from the followingdetailed description. It should be understood, however, that thedetailed description and the specific examples, while indicatingspecific embodiments, are given by way of illustration only, sincevarious changes and modifications within the spirit and scope of theinstant disclosure will become apparent to those skilled in the art fromthis detailed description.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference for the specificpurposes identified herein.

DETAILED DESCRIPTION OF THE INVENTION

Compound A refers to(S)-1-(3-(5,6-dihydroxypyrimidin-4-yl)-2-(4-((4-(morpholinomethyl)phenyl)ethynyl)phenyl)propyl)azetidine-3-carbonitrilewhich has the chemical structure shown below.

Compound A is a potent inhibitor ofUDP-{3—O—[(R)-3-hydroxymyristoyl]}-N-acetylglucosamine deacetylase(LpxC). LpxC is an essential enzyme involved in the first committed stepin lipid A biosynthesis for gram-negative bacteria. Lipid A is anessential component of the outer membrane of gram-negative bacteria.LpxC is highly conserved across strains of gram-negative bacteria,making LpxC an attractive target to treat gram-negative infections.

Compound A is an LpxC inhibitor that is useful in the methods oftreatment described herein. In gram-negative bacterial cell lines,Compound A is a potent inhibitor, exhibiting MIC values of <1 μg/mLagainst E. coli and K. pneumoniae cell lines. Additionally, Compound Adoes not inhibit gram-positive bacterial cell lines, such as S. aureus.

The preparation and use of Compound A has been previously described(see, PCT/US2019/052021, which is incorporated by reference in itsentirety).

Preparation of Compounds

Compounds described herein are synthesized using standard synthetictechniques or using methods known in the art in combination with methodsdescribed herein. Unless otherwise indicated, conventional methods ofmass spectroscopy, NMR, HPLC are employed.

Compounds are prepared using standard organic chemistry techniques suchas those described in, for example, March's Advanced Organic Chemistry,6^(th) Edition, John Wiley and Sons, Inc. Alternative reactionconditions for the synthetic transformations described herein may beemployed such as variation of solvent, reaction temperature, reactiontime, as well as different chemical reagents and other reactionconditions.

In the reactions described, it may be necessary to protect reactivefunctional groups, for example hydroxy or amino groups, where these aredesired in the final product, in order to avoid their unwantedparticipation in reactions. A detailed description of techniquesapplicable to the creation of protecting groups and their removal aredescribed in Greene and Wuts, Protective Groups in Organic Synthesis,3rd Ed., John Wiley & Sons, New York, NY, 1999, and Kocienski,Protective Groups, Thieme Verlag, New York, NY, 1994, which areincorporated herein by reference for such disclosure).

Preparation of Compound A

Disclosed herein are novel methods for the synthesis of Compound A.

In some embodiments, Compound A is synthesized as outlined in Schemes1-4.

Briefly, in some embodiments, a compound of Formula 1 is esterified toyield a compound of Formula 2. In some embodiments, the compound ofFormula 2 is treated with diethyl oxalate to provide a compound ofFormula 3. In some embodiments, cyclization of the compound of Formula 3with formamidine acetate provides a compound of Formula 4. In domeembodiments, halogenation of the compound of Formula 4 provides acompound of Formula 5. In some embodiments, treatment of the aryl halidewith a suitable alcohol (PG-OH) yields a compound of Formula 6, or acompound of Formula 7, or a combination thereof. In other embodiments, acompound of Formula 4 is treated with a suitable reagent to yield theprotected compound of Formula 6 directly. In some embodiments, thecompound of Formula 6 or Formula 7, or the combination thereof, isconverted to the compound of Formula 8 by reduction of the ester.Finally, in some embodiments, the alcohol of Formula 8 is converted to aleaving group to yield the compound of Formula 9.

Briefly, in some embodiments, the chiral oxazolidinone of Formula 10 istreated with the acid of Formula 11 to yield the compound of Formula 12.In some embodiments, the ketone of Formula 12 is reacted with a compoundof Formula 9 (vide supra) to form the compound of Formula 13. In someembodiments, the oxazolidinone is cleaved under reducing conditions toyield the primary alcohol of Formula 14.

Briefly, in some embodiments, the compound of Formula 18, is reactedwith a compound of Formula 19, or a salt thereof, under reductiveamination conditions to yield the compound of Formula 20, or a saltthereof.

Briefly, in some embodiments, the primary alcohol compound of Formula 14is oxidized to the aldehyde containing compound of Formula 15. In someembodiments, the compound of Formula 15 is reacted with a compound ofFormula 16, or a salt thereof, under reductive amination conditions toyield the compound of Formula 17. In some embodiments, the compound ofFormula 17 is reacted with the compound of Formula 20 (vide supra) undercross-coupling conditions to yield the compound of Formula 21. Finally,in some embodiments, the protected compound of Formula 21 is treatedwith appropriate deprotection conditions to yield Compound A.

As disclosed herein, variables in Schemes 1-4 are defined as follows:each PG is a suitable protecting group; R is C₁-C₁₀ alkyl; X is Cl orBr; LG is a suitable leaving group; R₁ is C₁-C₁₀ alkyl, aryl, or benzyl;and X is halogen, —OTf, —OTs, or —OMs.

In some embodiments, each PG is the same suitable protecting group. Insome embodiments, each PG is a different suitable protecting group. Insome embodiments, each —O-PG is an ether, a benzyl ether, an acetal, ora silyl ether; wherein each PG is the same protecting group. In someembodiments, each —O-PG is a benzyl ether, an acetal, or a silyl ether;wherein each PG is the same protecting group. In some embodiments, eachPG is methyl, benzyl, p-methoxybenzyl, methoxymethyl,[2-(trimethylsilyl)ethoxy]methyl, triisopropylsilyl, ortert-butyldimethylsilyl. In some embodiments, each PG is benzyl.

In some embodiments, R is methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, t-butyl, pentyl, iso-amyl, hexyl, heptyl, octyl,or nonyl. In some embodiments, R is methyl, ethyl, or isopropyl. In someembodiments, R is methyl or ethyl. In some embodiments, R is ethyl.

In some embodiments, X′ is Cl. In other embodiments, X′ is Br.

In some embodiments, LG is a halogen, a sulfonate, or a sulfate. In someembodiments, LG is Cl, Br, I, mesylate, tosylate, or triflate. In someembodiments, LG is Cl, Br, I, —OTf, —OTs, or —OMs. In some embodiments,is a halogen. In some embodiments, LG is Cl, Br, or I. In someembodiments, LG is Br or I. In some embodiments, LG is Br.

In some embodiments, R₁ is C₁-C₆ alkyl, phenyl, naphthyl, or benzyl. Insome embodiments, R₁ is methyl, isopropyl, tert-butyl, phenyl, orbenzyl. In some embodiments, R₁ is isopropyl, phenyl, or benzyl. In someembodiments, R₁ is benzyl.

In some embodiments, X is halogen. In some embodiments, X is Cl, Br, orI. In some embodiments, X is Br or I. In some embodiments, X is Br. Insome embodiments, X is I. In some embodiments, X is —OTf, —OTs, or —OMs.

Step 1: Synthesis of a Compound of Formula 2

In some embodiments, a compound of Formula 1 is esterified to a compoundof Formula 2. In some embodiments, the esterification proceeds in asuitable alcohol solvent in the presence of a suitable acid. In someembodiments, the suitable solvent has the formula R—OH, wherein R isC₁-C₁₀ alkyl. In some embodiments, the suitable solvent is methanol,ethanol, or isopropanol. In some embodiments, the suitable solvent isethanol, and R is ethyl. In some embodiments, the suitable acid is aninorganic acid. In some embodiments, the suitable acid is HCl, HBr,HNO₃, or H₂SO₄, or the like. In some embodiments, the suitable acid isH₂SO₄. In some embodiments, the reaction is performed at an elevatedtemperature. In some embodiments, the reaction is performed at thereflux temperature of the reaction mixture. In some embodiments, thereaction is performed at the boiling point of the solvent used. In someembodiments, the solvent is ethanol, and the reaction is performed atabout 78-80° C.

In some embodiments, the compound of Formula 1 is Compound 1A:

In some embodiments, the compound of Formula 2 is Compound 2A:

Step 2: Synthesis of a Compound of Formula 3

In some embodiments, a compound of Formula 2 is treated with diethyloxalate in the presence of a suitable base and in a suitable solvent toyield the compound of Formula 3. In some embodiments, the suitable baseis sodium hydride, sodium methoxide, sodium ethoxide, lithium aluminumhydride, n-butyllithium, or the like. In some embodiments, the suitablebase is sodium hydride. In some embodiments, the sodium hydride isprovided as a 60% suspension in mineral oil. In some embodiments, thesuitable solvent is methanol, ethanol, dimethoxyethane,2-methyltetrahydrofuran, methyl tert-butyl ether, cyclopentyl methylether, tetrahydrofuran, diethyl ether, diisopropyl ether, 1,4-dioxane,or a combination thereof. In some embodiments, the suitable solvent isdimethoxyethane, 2-methyltetrahydrofuran, methyl tert-butyl ether,cyclopentyl methyl ether, tetrahydrofuran, diethyl ether, diisopropylether, 1,4-dioxane, or a combination thereof. In some embodiments, thesuitable solvent is tetrahydrofuran. In some embodiments, the reactionis performed at a low temperature. In some embodiments, the reaction isperformed at about 0° C. In some embodiments, the reaction mixture isallowed to warm to room temperature (about 25° C.).

In some embodiments, the compound of Formula 3 is Compound 3A:

In some embodiments, the compound of Formula 3 is not isolated, and istaken on to Step 3 directly. In some embodiments, after warming to roomtemperature and the reaction of Step 2 is complete, the reaction mixtureis cooled again and taken on to Step 3 directly.

Step 3: Synthesis of a Compound of Formula 4

In some embodiments, cyclization of the compound of Formula 3 withformamidine acetate in the presence of a suitable base and in a suitablesolvent provides a compound of Formula 4. In some embodiments, thesuitable base is sodium hydride, sodium methoxide, sodium ethoxide,lithium methoxide, lithium ethoxide, n-butyl lithium, lithiumdiisopropylamide (LDA), lithium bis(trimethylsilyl)amide (LiHMDS), orlithium tetramethylpiperidide (LiTMP), or the like. In some embodiments,the suitable base is sodium methoxide, sodium ethoxide, lithiummethoxide, lithium ethoxide, or the like. In some embodiments, thesuitable base is sodium ethoxide. In some embodiments, the suitablesolvent is methanol, ethanol, isopropyl alcohol, diethyl ether,diisopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran,1,4-dioxane, tert-butyl methyl ether, cyclopropyl methyl ether, or acombination thereof. In some embodiments, the suitable solvent ismethanol or ethanol. In some embodiments, the suitable solvent isethanol. In some embodiments, the reaction is performed at a lowtemperature. In some embodiments, the reaction is performed at 0° C. Insome embodiments, the reaction is performed at a temperature of about0-10° C.

In some embodiments, when the compound of Formula 3 is not isolated fromStep 2 the suitable solvent is a mixture of methanol or ethanol andtetrahydrofuran. In some embodiments, the suitable solvent is a mixtureof ethanol and tetrahydrofuran. In some embodiments, the suitable baseis sodium ethoxide and the suitable solvent is a mixture of ethanol andtetrahydrofuran. In some embodiments, the reaction mixture from Step 2is cooled to a temperature of about 0-10° C. and the reaction of Step 3is performed.

In some embodiments, the compound of Formula 4 is Compound 4A:

Step 4: Synthesis of a Compound of Formula 5

In some embodiments, the hydroxyl group of the compound of Formula 4 isconverted to a halogen atom (X′ is Br or Cl), providing the compound ofFormula 5, by a halogenating agent and a suitable base in a suitablesolvent. In some embodiments, halogenation is bromination. In someembodiments, halogenation is chlorination. In some embodiments, thehalogenating agent is phosphorus oxychloride (POCl₃), thionyl chloride(SOCl₂), phosphorus tribromide (PBr₃), phosphorus oxybromide (POBr₃),hydrobromic acid, bromine, dibromotriphenylphosphorane, or the like. Insome embodiments, halogenation is chlorination and the halogenatingagent is phosphorus oxychloride (POCl₃) or thionyl chloride (SOCl₂). Insome embodiments, halogenation is bromination and the halogenating agentis phosphorus tribromide (PBr₃), phosphorus oxybromide (POBr₃),hydrobromic acid, bromine, or dibromotriphenylphosphorane. In someembodiments, the halogenating agent is POCl₃, POBr₃, or SOCl₂. In someembodiments, the halogenating agent is POCl₃. In some embodiments, thesuitable base is triethylamine, diisopropylethylamine, sec-butylamine,1,2,2,6,6-pentamethylpiperidine, tributylamine, or1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), or the like. In someembodiments, the suitable base is triethylamine. In some embodiments,the suitable solvent is acetonitrile, water, ethanol, isopropanol,dichloromethane, chloroform, dichloroethane, toluene,N,N-dimethylformamide, acetic acid, acetone, or the like. In someembodiments, the suitable solvent is acetonitrile, dichloromethane,chloroform, dichloroethane, toluene, or a combination thereof. In someembodiments, the suitable solvent is toluene. In some embodiments, thereaction is performed at an elevated temperature. In some embodiments,the reaction is performed at the reflux temperature of the reactionmixture. In some embodiments, the reaction is performed at about 85-95°C.

In some embodiments, the compound of Formula 5 is Compound 5A:

Step 5: Synthesis of a Compound of Formula 6, Formula 7, or aCombination Thereof

In some embodiments, treatment of the aryl halide of Formula 5 with asuitable alcohol (PG-OH) yields a compound of Formula 6, or a compoundof Formula 7, or a combination thereof. In some embodiment, the reactionyields a compound of Formula 6. In other embodiments, the reactionyields a compound of Formula 7. In other embodiments, the reactionyields a combination of a compound of Formula 6 and a compound ofFormula 7. In such embodiments, —O-PG is an ether or a benzyl ether.

In some embodiments, the suitable alcohol (R—OH) is methanol, ethanol,or benzyl alcohol. In some embodiments, the suitable alcohol (R—OH) isbenzyl alcohol. In some such embodiments, the compound of Formula 6 is acompound of Formula 6-I, and the compound of Formula 7 is a compound ofFormula 7-1:

In some embodiments, the reaction comprises a suitable base and is runin a suitable solvent. In some embodiments, the suitable base istriethylamine, diisopropylethylamine, sec-butylamine,1,2,2,6,6-pentamethylpiperidine, tributylamine, or1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), or the like. In someembodiments, the suitable base is triethylamine, diisopropylethylamine,1,2,2,6,6-pentamethylpiperidine, tributylamine, or1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). In some embodiments, thesuitable base is DBU. In some embodiments, the suitable solvent isacetonitrile, dimethylformamide, diethyl ether, ethanol,tetrahydrofuran, isopropyl alcohol, 1,4-dioxane, toluene, water, or acombination thereof. In some embodiments, the suitable solvent isacetonitrile. In other embodiments, the suitable solvent is the suitablealcohol having formula R—OH. In some such embodiments, the suitablesolvent is methanol, ethanol, or benzyl alcohol.

In some embodiments, the compound of Formula 6 or Formula 6-1 isCompound 6A:

In some embodiments, the compound of Formula 7 or Formula 7-I isCompound 7A:

Step Alt-5: Alternative Synthesis of a Compound of Formula 6

In other embodiments, a compound of Formula 4 is treated with a suitablereagent in a suitable solvent to yield the protected compound of Formula6 directly.

In some such embodiments, the suitable reagent is a benzyl halide, suchas a benzyl bromide. In such embodiments, —O-PG is a benzyl ether. Insome such embodiments, the suitable reagent is benzyl bromide,p-methoxybenzyl bromide, or the like. In such embodiments, PG is benzyl,p-methoxybenzyl, respectively, or the like. In some embodiments, thesuitable solvent is acetonitrile, dichloromethane, dimethylformamide,acetone, diethyl ether, ethanol, tetrahydrofuran, isopropyl alcohol,1,4-dioxane, toluene, water, or a combination thereof.

In other such embodiments, the suitable reagent is a silyl chloride. Insuch embodiments, —O-PG is a silyl ether. In some such embodiments, thesuitable reagent is triisopropylsilyl chloride ortert-butyldimethylsilyl chloride, or the like. In such embodiments, PGis triisopropylsilyl or tert-butyldimethylsilyl, respectively, or thelike. In some embodiments, the suitable base is imidazole. In someembodiments, the suitable solvent is acetonitrile, dichloromethane,dimethylformamide, diethyl ether, ethanol, tetrahydrofuran, isopropylalcohol, 1,4-dioxane, toluene, water, or a combination thereof.

In other such embodiments, the suitable reagent is a chloromethyl ether.In such embodiments, —O-PG is an acetal. In some such embodiments, thesuitable reagent is [2-(trimethylsilyl)ethoxy]methyl chloride orchloromethyl methyl ether, or the like. In such embodiments, PG is[2-(trimethylsilyl)ethoxy]methyl or methoxymethyl, respectively, or thelike. In some embodiments, the suitable base is triethylamine,diisopropylethylamine, 1,2,2,6,6-pentamethylpiperidine, tributylamine,or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), or the like. In someembodiments, the suitable solvent is acetonitrile, dichloromethane,dimethylformamide, diethyl ether, ethanol, tetrahydrofuran, isopropylalcohol, 1,4-dioxane, toluene, water, or a combination thereof.

Step 6: Synthesis of a Compound of Formula 8

In some embodiments, the compound of Formula 6 or Formula 7, or thecombination thereof, is converted to the compound of Formula 8 byreduction of the ester with suitable reducing agent in a suitablesolvent. In some embodiments, the suitable reducing agent is aborohydride. In some embodiments, the suitable reducing agent is sodiumborohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, orlithium cyanoborohydride, or the like. In some embodiments, the suitablereducing agent is sodium borohydride. In some embodiments, the suitablesolvent is acetonitrile, dimethylformamide, diethyl ether, methanol,ethanol, tetrahydrofuran, isopropyl alcohol, 1,4-dioxane, toluene,water, or a combination thereof. In some embodiments, the suitablesolvent is methanol, ethanol, isopropyl alcohol, water, or a combinationthereof. In some embodiments, the suitable solvent is a mixture ofisopropyl alcohol and methanol. In some embodiments, the reaction isperformed at a low temperature. In some embodiments, the reaction isperformed at about 0° C. In some embodiments, the reaction mixture isallowed to warm to room temperature (about 25° C.).

In embodiments where the compound of Formula 6 or Formula 7 is acompound of Formula 6-I or 7-I, the compound of Formula 8 is a compoundof Formula 8-I:

In some embodiments, the compound of Formula 8 or Formula 8-1 isCompound 8A:

Step 7: Synthesis of a Compound of Formula 9

In some embodiments, the alcohol —OH group of Formula 8 is converted toa leaving group to yield the compound of Formula 9, by treatment with asuitable reagent in a suitable solvent. In some embodiments, thesuitable reagent is a halogenating agent, a sulfonating agent, or asulfonyl chloride. In some embodiments, the suitable reagent is ahalogenating agent. In such embodiments, LG is a halogen. In someembodiments, LG is Cl, Br, or I. In some embodiments, LG is Br or I. Insome embodiments, LG is Cl or Br. In some embodiments, LG is Br. In someembodiments, the suitable reagent is SOCl₂, PBr₃, or PCl₃, or the like.In some embodiments, the suitable reagent is PBr₃. In some embodiments,the suitable reagent is a sulfonating agent. In such embodiments, LG isa sulfate. In some embodiments, the suitable reagent is a sulfonylchloride. In such embodiments, LG is a sulfonate. In some embodiments,the suitable reagent is tosyl chloride, mesyl chloride, or triflylchloride, or the like. In such embodiments, LG is a tosylate, mesylate,or triflate, respectively, or the like. In some embodiments, thesuitable solvent is acetonitrile, dimethylformamide, diethyl ether,ethanol, tetrahydrofuran, isopropyl alcohol, 1,4-dioxane, toluene,water, or a combination thereof. In some embodiments, the suitablesolvent is dimethylformamide. In some embodiments, the reaction isperformed at about 0° C.

In some embodiments, Step 7 further comprises a suitable base. In someembodiments, the suitable base is pyridine, N-methylmorpholine,triethylamine, diisopropylethylamine, sec-butylamine,1,2,2,6,6-pentamethylpiperidine, tributylamine, and1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), or the like. In someembodiments, the suitable base is pyridine.

In embodiments where the compound of Formula 8 is a compound of Formula8-I, the compound of Formula 9 is a compound of Formula 9-1:

In some embodiments, the compound of Formula 9 or Formula 9-1 isCompound 9A:

Step 8: Synthesis of a Compound of Formula 12

In some embodiments, using standard oxazolidinone chiral auxiliarychemistry, the chiral oxazolidinone of Formula 10 is treated with theacid of Formula 11 to yield the amide of Formula 12. In someembodiments, the amide formation proceeds with a suitable reagent, asuitable base, and in a suitable solvent. In some embodiments, thesuitable reagent is BOP, PyBOP, HATU, HBTU, pivaloyl chloride, or thelike. In some embodiments, the suitable reagent is pivaloyl chloride. Insome embodiments, the use of pivaloyl chloride is more cost-effectivethan other expensive amide coupling reagents. In some embodiments, thesuitable base is N-methylmorpholine, triethylamine,diisopropylethylamine, sec-butylamine, 1,2,2,6,6-pentamethylpiperidine,tributylamine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), or thelike. In some embodiments, the suitable base is N-methylmorpholine. Insome embodiments, the suitable solvent is acetonitrile,dimethylformamide, diethyl ether, ethanol, tetrahydrofuran, isopropylalcohol, 1,4-dioxane, toluene, or a combination thereof. In someembodiments, the suitable solvent is toluene. In some embodiments, thereaction is performed at elevated temperature. In some embodiments, thereaction is performed at the reflux temperature of the reaction mixture.In some embodiments, the reaction is performed at the boiling point ofthe solvent used. In some embodiments, the solvent is toluene, and thereaction is performed at about 107-112° C.

In some embodiments, the compound of Formula 10 is Compound 10A:

In some embodiments, the compound of Formula 11 is Compound 11A:

In some embodiments, the compound of Formula 12 is Compound 12A:

Step 9: Synthesis of a Compound of Formula 13

In some embodiments, using standard oxazolidinone chiral auxiliarychemistry, the chiral oxazolidinone amide of Formula 12 is reacted witha suitable base and the compound of Formula 9 (vide supra) in a suitablesolvent to provide the compound of Formula 13. In some embodiments, thesuitable base is n-butyl lithium, lithium diisopropylamide (LDA),lithium bis(trimethylsilyl)amide (LiHMDS), or lithiumtetramethylpiperidide (LiTMP), or the like. In some embodiments, thesuitable base is LiHMDS. In some embodiments, the suitable solvent isdiethyl ether, diisopropyl ether, tetrahydrofuran,2-methyltetrahydrofuran, 1,4-dioxane, tert-butyl methyl ether,cyclopropyl methyl ether, or a combination thereof. In some embodiments,the suitable solvent is tetrahydrofuran. In some embodiments, thereaction is performed at a temperature below 0° C. In some embodiments,the reaction is performed at a temperature of about −25 to about −15° C.

In some embodiments, the compound of Formula 12 is reacted with thesuitable base before addition of the compound of Formula 9. In someembodiments, the compound of Formula 12 is reacted with the base forfrom about 15 min to about 60 min before the addition of the compound ofFormula 9. In some embodiments, the compound of Formula 12 is reactedwith the base for from about 15 min before the addition of the compoundof Formula 9.

In some embodiments, the compound of Formula 13 is Compound 13A:

In some embodiments, the process of Steps 8 and 9 produces a singlediastereomer of a compound of Formula 13 (such as Compound 13A). In someembodiments, the process produces >95% of a single diastereomer of acompound of Formula 13 (such as Compound 13A). In some embodiments,isolation of the compound of Formula 13 (such as Compound 13A) comprisesa crystallization step. In some embodiments, the process of Steps 8 and9 produces >95%, >96%, >97%, >98%, or >99% of a single diastereomer of acompound of Formula 13 (such as Compound 13A). In some embodiments, thecompound of Formula 13 (such as Compound 13A) is isolated with 99% de,99.1% de, 99.2% de, 99.3% de, 99.4% de, 99.5% de, or >99.5% de.

Step 10: Synthesis of a Compound of Formula 14

In some embodiments, the oxazolidinone is cleaved under reducingconditions to yield the primary alcohol of Formula 14. In someembodiments, the compound of Formula 13 is treated with a suitableborohydride reagent in a suitable solvent to provide the compound ofFormula 14. In some embodiments, the suitable borohydride reagent islithium borohydride, sodium borohydride, sodium cyanoborohydride,potassium borohydride, lithium triethylborohydride, or sodiumtriacetoxyborohydride, or the like. In some embodiments, the suitableborohydride reagent is lithium borohydride. In some embodiments, thesuitable solvent is acetonitrile, methanol, ethanol, isopropyl alcohol,dimethoxyethane, 2-methyltetrahydrofuran, methyl tert-butyl ether,cyclopentyl methyl ether, tetrahydrofuran, diethyl ether, diisopropylether, 1,4-dioxane, toluene, water, or a combination thereof. In someembodiments, the suitable solvent is methanol, ethanol, isopropylalcohol, tetrahydrofuran, 1,4-dioxane, or a combination thereof. In someembodiments, the suitable solvent is a mixture of tetrahydrofuran andethanol. In some embodiments, the reaction is performed at a lowtemperature. In some embodiments, the reaction is performed at about 0°C. In some embodiments, the reaction mixture is allowed to warm to roomtemperature (about 25° C.).

In some embodiments, the compound of Formula 14 is Compound 14A:

Step 10-A: Crystallization of a Compound of Formula 14

In some embodiments, the compound of Formula 14 (such as Compound 14A)is recrystallized. In some embodiments, the compound of Formula 14 (suchas Compound 14A) is recrystallized from acetonitrile, methanol, ethanol,isopropyl alcohol, acetone, methyl acetate, ethyl acetate,dichloromethane, chloroform, diethyl ether, diisopropyl ether,tert-butyl methyl ether, tetrahydrofuran, 2-methyltetrahydrofuran,dioxane, benzene, toluene, petroleum ether, pentane, hexane, heptane,cyclohexane, acetic acid, water, or a mixture thereof. In someembodiments, the compound of Formula 14 (such as Compound 14A) isrecrystallized from methanol, ethanol, isopropyl alcohol, or water, or amixture thereof. In some embodiments, the compound of Formula 14 (suchas Compound 14A) is recrystallized from a mixture of isopropyl alcoholand water. In some embodiments, the compound of Formula 14 (such asCompound 14A) is recrystallized from a mixture of isopropanol and waterin a ratio of about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, orabout 1:2 isopropyl alcohol to water.

In some embodiments, the process of Steps 8-10 produces a singleenantiomer of a compound of Formula 14 (such as Compound 14A). In someembodiments, the process produces >95% of a single enantiomer of acompound of Formula 14 (such as Compound 14A). In some embodiments, theprocess of Steps 8-10 produces >95%, >96%, >97%, >98%, or >99% of asingle enantiomer of a compound of Formula 14 (such as Compound 14A). Insome embodiments, the compound of Formula 14 (such as Compound 14A) isisolated with 99% ee, 99.1% ee, 99.2% ee, 99.3% ee, 99.4% ee, 99.5% ee,99.6% ee, 99.7% ee, 99.8% ee, 99.9% ee, or >99.9% ee.

Step 11: Synthesis of a Compound of Formula 20, or a Salt Thereof

In some embodiments, the aldehyde of Formula 18 and morpholine (thecompound of Formula 19), or a salt thereof, are treated with a suitableborohydride reagent in a suitable solvent to yield the compound ofFormula 20, or a salt thereof. In some embodiments, the suitableborohydride reagent is lithium borohydride, sodium borohydride, sodiumcyanoborohydride, potassium borohydride, lithium triethylborohydride, orsodium triacetoxyborohydride, or the like. In some embodiments, thesuitable borohydride reagent is sodium triacetoxyborohydride. In someembodiments, the suitable solvent is acetonitrile, methanol, ethanol,isopropyl alcohol, dimethoxyethane, 2-methyltetrahydrofuran, methyltert-butyl ether, cyclopentyl methyl ether, tetrahydrofuran, diethylether, diisopropyl ether, 1,4-dioxane, toluene, water, or a combinationthereof. In some embodiments, the suitable solvent is methanol, ethanol,isopropyl alcohol, tetrahydrofuran, 1,4-dioxane, or a combinationthereof. In some embodiments, the suitable solvent is tetrahydrofuran.In some embodiments, the reaction is performed at a low temperature. Insome embodiments, the reaction is performed at about 0° C. In someembodiments, the reaction mixture is allowed to warm to room temperature(about 25° C.).

In some embodiments, the compound of Formula 20 is isolated as a salt.In some embodiments, the compound of Formula 20 is isolated the HCladdition salt. In some embodiments, the compound of Formula 20, or saltthereof, is Compound 20A:

Step 12: Synthesis of a Compound of Formula 15

In some embodiments, the primary alcohol compound of Formula 14 istreated with a suitable oxidation reagent system in a suitable solventto provide the aldehyde compound of Formula 15. In some embodiments, thesuitable oxidation reagent system is a chromium based reagent (such asCollins reagent, pyridinium dichromate, or pyridinium chlorochromate), asulfonium species, a hypervalent iodine reagent (such as Dess-Martinperiodinane or 2-iodobenzoic acid), a TPAP/NMO system, or a TEMPO/bleachsystem. In some embodiments, the suitable solvent is acetonitrile,dimethylsulfoxide, dichloromethane, chloroform, dichloroethane, hexanes,ethyl acetate, acetic acid, toluene, water, or a combination thereof.

In preferred embodiments, the suitable oxidation reagent system is aTEMPO/bleach system. In some embodiments, the reaction mixture comprisessubstoichiometric or a catalytic amount of TEMPO. In some embodiments,the reaction mixture comprises about 0.0025, 0.005, 0.0075, 0.01, 0.015,0.02, 0.025, 0.03, 0.05, 0.075, or 0.10 equivalents of TEMPO. In someembodiments, the reaction mixture comprises about 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.75, or 2.0 equivalents of bleach. In some embodiments, thepH of the bleach solution is adjusted. In some embodiments, the pH isadjusted to about 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5. In some embodiments,the pH is adjusted with NaHCO₃. In some embodiments, the reactionmixture further comprises a suitable salt. In some embodiments, thesuitable salt is KBr. In some embodiments, the suitable solvent isacetonitrile, dichloromethane, chloroform, dichloroethane, hexanes,water, or a combination thereof. In some embodiments, the suitablesolvent is a mixture of water and dichloromethane. In some embodiments,the reaction is performed at about 0° C. In some embodiments, thereaction mixture is performed at about 0-5° C. In some embodiments, thereaction is kept below 5° C.

In some embodiments, the compound of Formula 15, or salt thereof, isCompound 15A.

In some embodiments, using a TEMPO/bleach oxidation system does notdegrade the stereochemistry of the compound. In some embodiments, usinga TEMPO/bleach oxidation system does not significantly degrade thestereochemistry of the compound. In some embodiments, the ee of thecompound of Formula 15 (such as Compound 15A) is the same as the ee ofthe compound of Formula 14 (such as 14A). In some embodiments, the ee ofthe compound of Formula 15 (such as Compound 15A) is substantially thesame as the ee of the compound of Formula 14 (such as 14A). In someembodiments, the ee of the compound of Formula 15 (such as Compound 15A)is +0.2 or 0.1 of the ee of the compound of Formula 14 (such as 14A).

In some embodiments, the compound of Formula 15 is not isolated, and istaken on to Step 13 directly. In some embodiments, after workup of thereaction of Step 12, the compound of Formula 15 is not isolated, and istaken on to Step 13 directly. In some embodiments, the water later isremoved from the reaction mixture of Step 12, and the organic layer istaken onto Step 13 directly without isolation of the compound of Formula15 (such as Compound 15A).

Step 13: Synthesis of a Compound of Formula 17

In some embodiments, the aldehyde of Formula 15 and the compound ofFormula 16, or a salt thereof, are treated with a suitable reducingagent in a suitable solvent to yield the compound of Formula 17, or asalt thereof. In some embodiments, the suitable reducing agent is sodiumborohydride, sodium cyanoborohydride, sodium triacetoxyborohydride,lithium cyanoborohydride, H₂/catalyst, or picoline-borane. In someembodiments, the suitable reducing agent is sodium borohydride, sodiumcyanoborohydride, sodium triacetoxyborohydride, lithiumcyanoborohydride, or picoline-borane. In some embodiments, the suitablereducing agent is a borohydride reagent or picoline-borane. In someembodiments, the suitable reducing agent is picoline-borane. In someembodiments, the suitable solvent is acetonitrile, methanol, ethanol,dichloromethane, chloroform, dichloroethane, toluene, water, or acombination thereof. In some embodiments, the suitable solvent is amixture of methanol and dichloromethane. In some embodiments, thereaction is performed at about 0° C. In some embodiments, the reactionmixture is allowed to warm to room temperature (about 25° C.).

In some embodiments, when the compound of Formula 15 is not isolatedfrom Step 12, the suitable solvent is a mixture of dichloromethane andmethanol. In some embodiments, the reaction mixture from Step 2 ismaintained at a temperature of about 0-5° C. and the reaction of Step 13is performed. In some embodiments, the reaction is performed at about 0°C. In some embodiments, the reaction mixture is allowed to warm to roomtemperature (about 25° C.).

In some embodiments, the compound of Formula 16 is used as a salt. Insome embodiments, the compound of Formula 16, or salt thereof, isCompound 16A:

In some embodiments, the compound of Formula 17, or salt thereof, isCompound 17A:

In some embodiments, the compound of Formula 17 (such as Compound 17A)is isolated as the HCl addition salt. In such embodiments, the compoundof Formula 17 (such as Compound 17A) is converted to the free base forisolation and purification. In some embodiments, the compound of Formula17 (such as Compound 17A) is isolated as the free base after aqueousextractive workup.

Step 14: Synthesis of a compound of Formula 21

In some embodiments, the compound of Formula 17 is reacted with thecompound of Formula 20 in the presence of a coupling catalyst, asuitable base, and in a suitable solvent to provide a compound ofFormula 21.

In some embodiments, the coupling catalyst is a palladium catalyst. Insome embodiments, the palladium catalyst is a palladium(0) catalyst. Inother embodiments, the palladium catalyst is a palladium(II) catalyst.In some embodiments, the palladium catalyst is precoordinated with aligand. In some embodiments, the palladium catalyst is Pd(PPh₃)₂Cl₂. Insome embodiments, the palladium catalyst is Pd(PPh₃)₃Cl. In someembodiments, the palladium catalyst is Pd(PPh₃)₄. In some embodiments,the amount of palladium used is from about 0.005 equiv to about 0.1equiv. In some embodiments, the amount of palladium used is about 0.005,0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 equiv. Insome embodiments, the amount of palladium used is about 0.02 equiv.

In some embodiments, Step 14 further comprises adding an exogenousligand. In some embodiments, the ligand is a phosphine ligand. In someembodiments, the ligand is an aliphatic phosphine ligand, such astrimethyl phosphine, tricyclohexylphosphine, tri-tert-butyl-phosphine orthe like. In some embodiments, the ligand is an aromatic phosphine, suchas XPhos, SPhos, JohnPhos, Amphos, triphenylphosphine,methyldiphenylphosphine, or the like. In some embodiments, the ligand isa phosphite ligand, such as trimethylphosphite, triphenylphosphite, orthe like. In some embodiments, the ligand is a bis-phosphine ligand,such as diphenylphosphinomethane (dppm), diphenyl phosphinoethane(dppe), 1,1′-bis(diphenylphosphino)ferrocene (dppf), or the like. Insome embodiments, the ligand is triphenylphospine.

In some embodiments, Step 14 further comprises adding a copper(I)cocatalyst. In some embodiments, the copper(I) cocatalyst in Step 1 is acopper(I) salt. In some embodiments, the copper(I) cocatalyst in Step 1is CuCl, CuBr, or CuI. In some embodiments, the copper(I) cocatalyst isCuI. In some embodiments, the copper(I) cocatalyst is acopper(I)—N-heterocyclic carbene (Copper-NHC) complex. In someembodiments, the amount of copper(I) cocatalyst used in Step 1 is fromabout 0.001 equiv to about 0.1 equiv. In some embodiments, the amount ofcopper(I) cocatalyst used in Step 1 is about 0.001, 0.002, 0.003, 0.004,0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1equiv. In some embodiments, the amount of copper(I) cocatalyst used inStep 1 is about 0.005 equiv.

In preferred embodiments, Step 13 does not comprise a copper(I)cocatalyst. In some instances, additional metals, such as copper,complicate the purification of the compounds described herein, orcontaminate the final product, Compound A. In such instances, it ispreferable to avoid the additional metal.

In some embodiments, the suitable base is an amine base. In someembodiments, the suitable base is a primary, a secondary, or a tertiaryamine base. In some embodiments, the suitable base is a quaternaryammonium salt. In some embodiments, the suitable base is triethylamine,diisopropylethylamine, 1,2,2,6,6-pentamethylpiperidine, tributylamine,1,8-diazabicycloundec-7-ene (DBU), or the like. In some embodiments, thesuitable base is sec-butylamine. In some embodiments, the suitable baseis tetrabutylammonium fluoride (TBAF).

In preferred embodiments, the suitable base is sec-butylamine ortetrabutylammonium fluoride (TBAF). Both of these bases allow thereaction to proceed without the addition of a copper co-catalyst. In amore preferred embodiment, the suitable base is sec-butylamine. Inpreferred embodiments, sec-butylamine is amenable to use in steelreaction vessels. In some embodiments, sec-butylamine is used in excess.In some embodiments, sec-butyl amine is used as a cosolvent.

In some embodiments, the suitable solvent is acetonitrile,dimethylformamide, diethyl ether, ethanol, tetrahydrofuran, isopropylalcohol, 1,4-dioxane, toluene, water, or a combination thereof. In someembodiments, the suitable solvent is water.

In preferred embodiments, sec-butylamine and water are used in a ratioof about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1 water tosec-butylamine.

In some embodiments, the compound of Formula 21, is Compound 21A:

Step 15: Synthesis of a Compound A

In some embodiments, the protected compound of Formula 21 is treatedwith appropriate deprotection conditions to yield Compound A. Theseconditions vary based on the nature of the protecting group(s) used. Insome embodiments, the protecting groups are the same. In otherembodiments the protecting groups are different. In some embodiments,the two protecting groups are removed in a single reaction. In otherembodiments, two successive reactions are required for completedeprotection of the two protecting groups.

In some embodiments, the compound of Formula 21 is treated with asuitable reagent in a suitable solvent to facilitate the deprotection.In some embodiments, the suitable reagent is H₂/catalyst, HCl, HBr, TFA,TBAF, BCl₃, 9-1-BBN, BF₃—OEt₂, TMS-Cl, or TMS-Br, or the like.

In some embodiments, the suitable solvent is acetonitrile,dichloromethane, chloroform, dichloroethane, diethyl ether,tetrahydrofuran, isopropyl alcohol, 1,4-dioxane, toluene, anisole,water, or a combination thereof.

In some embodiments, when —O-PG is a benzyl ether, the suitable reagentis H₂/catalyst, TFA, BCl₃, or 9-I-BBN, or the like.

In preferred embodiments, when PG is Bn, the suitable reagent is TFA. Insome such embodiments, Step 15 further comprises the addition ofpentamethylbenzene. In such embodiments, a TFA/pentamethylbenzenedeprotection provides higher yields and a cleaner reaction. In some suchembodiments, the suitable solvent is anisole.

In some embodiments, when —O-PG is an acetal (as in a methoxymethylgroup), the suitable reagent is HCl, HBr, or TMS-Br, or the like.

In some embodiments, when —O-PG is a silyl ether, or comprises a silylgroup and an ether (as in [2-(trimethylsilyl)ethoxy]methyl group), thesuitable reagent TBAF, or the like.

In some embodiments, the reaction is performed at a low temperature. Insome embodiments, the reaction is performed at about 0° C. In someembodiments, the reaction mixture is allowed to warm to room temperature(about 25° C.).

Step 15-A: Crystallization of Compound A

In some embodiments, Compound A is recrystallized. In some embodiments,Compound A is recrystallized from acetonitrile, methanol, ethanol,isopropyl alcohol, acetone, methyl acetate, ethyl acetate,dichloromethane, chloroform, diethyl ether, diisopropyl ether,tert-butyl methyl ether, tetrahydrofuran, 2-methyltetrahydrofuran,dioxane, benzene, toluene, petroleum ether, pentane, hexane, heptane,cyclohexane, acetic acid, water, or a mixture thereof.

In some embodiments, Compound A is recrystallized from a mixture ofacetic acid and tetrahydrofuran. In some embodiments, Compound A isrecrystallized from a mixture of acetic acid and tetrahydrofuran in aratio of about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about1:5, about 1:6, about 1:7, about 1:8, or about 1:9 acetic acid totetrahydrofuran.

In some instances, the compound of Formula 8 is synthesized as outlinedin Scheme 5.

Briefly, in some embodiments, the methyl ether of Formula 22 isdeprotected to yield a compound of Formula 23. In some embodiments, thecompound of Formula 23 is treated with a suitable reagent to yield theprotected compound of Formula 24. In some embodiments, treatment of thearyl halide 24 with a suitable alcohol (PG-OH) yields a compound ofFormula 25. In some embodiments, the compound of Formula 25 ismethylated under cross-coupling conditions with a compound having theformula CH₃—B, to yield the compound of Formula 26, wherein B is aboronic acid, boronate ester, or trifluoroborate. In some embodiments,the compound of Formula 26 is oxidized to yield a compound of Formula27. Finally, in some embodiments, the N-oxide compound of Formula 27undergoes a rearrangement to yield the compound of Formula 8.

As disclosed herein, variables in Scheme 5 are defined as follows: eachPG is a suitable protecting group; and B is a boronic acid, boronateester, or trifluoroborate.

In some embodiments, each PG is the same suitable protecting group. Insome embodiments, each PG is a different suitable protecting group. Insome embodiments, each —O-PG is an ether, a benzyl ether, an acetal, ora silyl ether; wherein each PG is the same protecting group. In someembodiments, each —O-PG is a benzyl ether, an acetal, or a silyl ether;wherein each PG is the same protecting group. In some embodiments, eachPG is methyl, benzyl, p-methoxybenzyl, methoxymethyl,[2-(trimethylsilyl)ethoxy]methyl, triisopropylsilyl, ortert-butyldimethylsilyl. In some embodiments, each PG is benzyl.

Step 1: Synthesis of a Compound of Formula 23

In some embodiments, the methyl ether of Formula 22 is deprotected bytreatment with a suitable reagent and in a suitable solvent to yield acompound of Formula 23. In some embodiments, the suitable reagent isBBr₃, pyridine-HCl, BCl₃, NaSEt, AlCl₃, or the like. In someembodiments, the suitable reagent is AlCl₃. In some embodiments, thesuitable solvent is dimethylformamide, diethyl ether, tetrahydrofuran,1,4-dioxane, toluene, dichloromethane, chloroform, dichloroethane, orthe like, or a combination thereof. In some embodiments, the suitablesolvent is dichloromethane, chloroform, or dichloroethane. In someembodiments, the suitable solvent is dichloroethane. In someembodiments, the reaction is performed at about 0° C. In someembodiments, the reaction mixture is allowed to warm to room temperature(about 25° C.). In some embodiments, the reaction begins at about 0° C.and is warmed to about 50° C.

In some embodiments, the compound of Formula 22 is Compound 22A:

In some embodiments, the compound of Formula 23 is Compound 23A:

Step 2: Synthesis of a Compound of Formula 24

In some embodiments, a compound of Formula 23 is treated with a suitablereagent in a suitable solvent to yield the protected compound of Formula24.

In some embodiments, the suitable reagent is a benzyl halide, such as abenzyl bromide, and a suitable base. In such embodiments, —O-PG is abenzyl ether. In some such embodiments, the suitable reagent is benzylbromide or p-methoxybenzyl bromide, or the like. In such embodiments, PGis benzyl or p-methoxybenzyl, respectively, or the like. In someembodiments, the suitable reagent is benzyl bromide. In someembodiments, the suitable base is Na₂CO₃, NaHCO₃, K₂CO₃, Li₂CO₃, Cs₂CO₃,or the like. In some embodiments, the suitable base is K₂CO₃. In someembodiments, the suitable solvent is acetonitrile, dichloromethane,dimethylformamide, acetone, diethyl ether, ethanol, tetrahydrofuran,isopropyl alcohol, 1,4-dioxane, toluene, water, or a combinationthereof. In some embodiments, the suitable solvent is acetone. In someembodiments, the reaction further comprises a phase transfer catalyst,such as tetrabutylammonium iodide (TBAI).

In other embodiments, the suitable reagent is a silyl chloride. In suchembodiments, —O-PG is a silyl ether. In some such embodiments, thesuitable reagent is triisopropylsilyl chloride ortert-butyldimethylsilyl chloride, or the like. In such embodiments, PGis triisopropylsilyl or tert-butyldimethylsilyl, respectively, or thelike. In some embodiments, the suitable base is imidazole. In someembodiments, the suitable solvent is acetonitrile, dichloromethane,dimethylformamide, diethyl ether, ethanol, tetrahydrofuran, isopropylalcohol, 1,4-dioxane, toluene, water, or a combination thereof.

In other embodiments, the suitable reagent is a chloromethyl ether. Insuch embodiments, —O-PG is an acetal. In some such embodiments, thesuitable reagent is [2-(trimethylsilyl)ethoxy]methyl chloride orchloromethyl methyl ether, or the like. In such embodiments, PG is[2-(trimethylsilyl)ethoxy]methyl or methoxymethyl, respectively, or thelike. In some embodiments, the suitable base is triethylamine,diisopropylethylamine, 1,2,2,6,6-pentamethylpiperidine, tributylamine,or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), or the like. In someembodiments, the suitable solvent is acetonitrile, dichloromethane,dimethylformamide, diethyl ether, ethanol, tetrahydrofuran, isopropylalcohol, 1,4-dioxane, toluene, water, or a combination thereof.

In some embodiments, the compound of Formula 24 is Compound 24A:

Step 3: Synthesis of a Compound of Formula 25

In some embodiments, treatment of the aryl halide of Formula 24 with asuitable alcohol (PG-OH) yields a compound of Formula 25. In suchembodiments, —O-PG is an ether or a benzyl ether.

In some embodiments, the suitable alcohol (R—OH) is methanol, ethanol,or benzyl alcohol. In some embodiments, the suitable alcohol (R—OH) isbenzyl alcohol. In some such embodiments, the compound of Formula 25 isa compound of Formula 25-I:

In some embodiments, the reaction comprises a suitable base and is runin a suitable solvent. In some embodiments, the suitable base is sodiumhydride, triethylamine, diisopropylethylamine, sec-butylamine,1,2,2,6,6-pentamethylpiperidine, tributylamine, or1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), or the like. In someembodiments, the suitable base is sodium hydride. In some embodiments,the suitable solvent is acetonitrile, dimethylformamide, diethyl ether,ethanol, tetrahydrofuran, isopropyl alcohol, 1,4-dioxane, toluene,water, or a combination thereof. In some embodiments, the suitablesolvent is tetrahydrofuran. In other embodiments, the suitable solventis the suitable alcohol having formula R—OH. In some such embodiments,the suitable solvent is methanol, ethanol, or benzyl alcohol.

In some embodiments, the compound of Formula 25 or Formula 25-I isCompound 6A:

Step 4: Synthesis of a compound of Formula 26

In some embodiments, the compound of Formula 25 is reacted with acompound of the formula CH₃—B in the presence of a coupling catalyst, asuitable base, and in a suitable solvent to provide a compound ofFormula 26, wherein B is a boronic acid, boronate ester, ortrifluoroborate.

In some embodiments, B is a boronic acid or a boronic ester. In someembodiments, B

In some embodiments, B is a boronic acid. In some embodiments, B is

In some embodiments, B is a boronic ester. In some embodiments, B is atrifluoroborate. In some embodiments, B is

In some embodiments, the coupling catalyst is a palladium catalyst. Insome embodiments, the palladium catalyst is a palladium(0) catalyst. Inother embodiments, the palladium catalyst is a palladium(II) catalyst.In some embodiments, the palladium catalyst is precoordinated with aligand. In some embodiments, the palladium catalyst is Pd(PPh₃)₂Cl₂. Insome embodiments, the palladium catalyst is Pd(PPh₃)₃Cl. In someembodiments, the palladium catalyst is Pd(PPh₃)₄. In some embodiments,the amount of palladium used is from about 0.005 equiv to about 0.1equiv. In some embodiments, the amount of palladium used is about 0.005,0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 equiv. Insome embodiments, the amount of palladium used is about 0.02 equiv.

In some embodiments, the suitable base is an amine base. In someembodiments, the suitable base is a tertiary amine base. In someembodiments, the suitable base is triethylamine, diisopropylethylamine,1,2,2,6,6-pentamethylpiperidine, tributylamine,1,8-diazabicycloundec-7-ene (DBU), or the like. In other embodiments,the suitable base is an inorganic base. In some embodiments, thesuitable base is NaHCO₃, NaOAc, KOAc, Ba(OH)₂, Li₂CO₃, Na₂CO₃, K₂CO₃,Cs₂CO₃, Na₃PO₄, K₃PO₄, CsF, or the like. In some embodiments, thesuitable base is KOAc.

In some embodiments, Step 4 further comprises adding an exogenousligand. In some embodiments, the ligand is a phosphine ligand. In someembodiments, the ligand is an aliphatic phosphine ligand, such astrimethyl phosphine, tricyclohexylphosphine, tri-tert-butyl-phosphine orthe like. In some embodiments, the ligand is an aromatic phosphine, suchas XPhos, SPhos, JohnPhos, Amphos, triphenylphosphine,methyldiphenylphosphine, or the like. In some embodiments, the ligand isa phosphite ligand, such as trimethylphosphite, triphenylphosphite, orthe like. In some embodiments, the ligand is a bis-phosphine ligand,such as diphenylphosphinomethane (dppm), diphenyl phosphinoethane(dppe), 1,1′-bis(diphenylphosphino)ferrocene (dppf), or the like. Insome embodiments, the ligand is triphenylphospine.

In some embodiments, the suitable solvent is acetonitrile,dimethylformamide, diethyl ether, ethanol, tetrahydrofuran, isopropylalcohol, 1,4-dioxane, toluene, water, or a combination thereof. In someembodiments, the suitable solvent is 1,4-dioxane.

In some embodiments, the reaction is performed at an elevatedtemperature. In some embodiments, the reaction is performed at about100° C.

In embodiments where the compound of Formula 25 is a compound of Formula25-I, the compound of Formula 26 is a compound of Formula 26-1:

In some embodiments, the compound of Formula 26 or Formula 26-I isCompound 26A:

Step 5: Synthesis of a Compound of Formula 27

In some embodiments, the compound of Formula 26 is treated with asuitable oxidation reagent in a suitable solvent to yield a compound ofFormula 27. In some embodiments, the suitable oxidation reagent is aperoxyacetic acid or a peroxybenzoic acid. In some embodiments, thesuitable oxidation reagent is peracetic acid, m-chloroperoxybenzoic acid(mCPBA), or the like. In some embodiments, the suitable oxidationreagent is mCPBA. In other embodiments, the suitable oxidation reagentis hydrogen peroxide. In some embodiments, the suitable solvent isdichloromethane, chloroform, dichloroethane, or the like, or acombination thereof. In some embodiments, the suitable solvent isdichloromethane.

In embodiments where the compound of Formula 26 is a compound of Formula26-1, the compound of Formula 27 is a compound of Formula 27-I:

In some embodiments, the compound of Formula 27 or Formula 27-I isCompound 27A:

Step 6: Synthesis of a Compound of Formula 8

In some embodiments, the compound of Formula 27 is treated with asuitable reagent in a suitable solvent to yield a compound of Formula 8.In some embodiments, the compound of Formula 27 undergoes a[3,3]-sigmatropic rearrangement when treated with a suitable reagent ina suitable solvent to yield a compound of Formula 8. In someembodiments, the suitable reagent is an acid anhydride. In someembodiments, the suitable reagent is acetic anhydride, trifluoroaceticanhydride (TFAA), or the like. In some embodiments, the suitable reagentis TFAA. In some embodiments, the suitable solvent is acetonitrile,dichloromethane, chloroform, dichloroethane, dimethylformamide, diethylether, tetrahydrofuran, 1,4-dioxane, toluene, or a combination thereof.In some embodiments, the suitable solvent is dichloromethane.

In embodiments where the compound of Formula 27 is a compound of Formula27-I, the compound of Formula 8 is a compound of Formula 8-I:

In some embodiments, the compound of Formula 8 or Formula 8-I isCompound 8A:

Due to the fact that the synthetic methods described above utilize atransition metal catalyst, purification steps are performed to reducethe amount of palladium in the product. Purification steps to reduce theamount of palladium in a product are conducted so that activepharmaceutical ingredients meet palladium specification guidelines.(“Guideline on the Specification Limits for Residues of Metal Catalysts”European Medicines Agency Pre-authorisation Evaluation of Medicines forHuman Use, London, January 2007, Doc. Ref. CPMP/SWP/QWP/4446/00 corr.).In some embodiments, purification steps to reduce the amount ofpalladium in a product includes, but is not limited to, treatment withsolid trimercaptotriazine (TMT), polystyrene-bound TMT, mercapto-porouspolystyrene-bound TMT, polystyrene-bound ethylenediamine, activatedcarbon, glass bead sponges, Smopex™, silica bound scavengers,thiol-derivatized silica gel, N-acetylcysteine, n-Bu₃P, crystallization,extraction, l-cysteine, n-Bu₃P/lactic acid (Garrett el al., Adv. Synth.Catal. 2004, 346, 889-900). In some embodiments, activated carbonincludes but is not limited to DARCO® KB-G, DARCO® KB-WJ. In one aspectactivated bound scavengers include but are not limited to

where

denotes silica gel. In some embodiments, the purification steps toreduce the amount of palladium include the use of activated carbon,derivatized silica gel (e.g., thiol derivatized silica gel), orcombinations thereof.

In some embodiments, the compound of Formula 21 (such as Compound 21A),the compound of Formula 26 (such as 26A), or Compound A is furthertreated with a metal scavenger to remove residual palladium. In someembodiments, the metal scavenger comprises SiO₂, charcoal, aqueoussolution of L-cysteine, a Silicycle metal scavenger, Si-thiol, SiliaBondDMT, SiliaBond Cysteine, or 3-mercaptopropyl ethyl sulfide silica. Insome embodiments, the scavenger loading (w/w) is about 1:3, about 1:2,or about 1:1.

In some of these embodiments, palladium levels are reduced to about 10ppm. In some of these embodiments, palladium levels are reducedsufficiently to be undetectable.

In some embodiments, the presence of residual heavy metal (e.g.palladium) impurities is determined by utilizing methods known in theart. In some embodiments, the presence of residual heavy metal (e.g.palladium) impurities is determined by the use of inductively coupledplasma mass spectrometry (ICP-MS). In some embodiments, the presence ofresidual heavy metal (e.g. palladium) impurities is determined by theuse of techniques described in U.S. Pharmacopeia General Chapter <231>Heavy Metals.

In some embodiments, compounds described herein are synthesized asoutlined in the Examples.

Definitions

Unless otherwise stated, the following terms used in this applicationhave the definitions given below. The use of the term “including” aswell as other forms, such as “include”, “includes,” and “included,” isnot limiting. The section headings used herein are for organizationalpurposes only and are not to be construed as limiting the subject matterdescribed.

The term “halo” or, alternatively, “halogen” or “halide” means fluoro,chloro, bromo or iodo. In some embodiments, halo is fluoro, chloro, orbromo.

The term “bond” or “single bond” refers to a chemical bond between twoatoms, or two moieties when the atoms joined by the bond are consideredto be part of larger substructure. In one aspect, when a group describedherein is a bond, the referenced group is absent thereby allowing a bondto be formed between the remaining identified groups.

The term “moiety” refers to a specific segment or functional group of amolecule. Chemical moieties are often recognized chemical entitiesembedded in or appended to a molecule.

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

The term “prodrug” is meant to indicate a compound that is, in someembodiments, converted under physiological conditions or by solvolysisto a biologically active compound described herein. Thus, the term“prodrug” refers to a precursor of a biologically active compound thatis pharmaceutically acceptable. A prodrug is typically inactive whenadministered to a subject, but is converted in vivo to an activecompound, for example, by hydrolysis. The prodrug compound often offersadvantages of solubility, tissue compatibility or delayed release in amammalian organism (see, e.g., Bundgard, H., Design of Prodrugs (1985),pp. 7 9, 21 24 (Elsevier, Amsterdam). A discussion of prodrugs isprovided in Higuchi, T., et al., “Pro drugs as Novel Delivery Systems,”A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in DrugDesign, ed. Edward B. Roche, American Pharmaceutical Association andPergamon Press, 1987. The term “prodrug” is also meant to include anycovalently bonded carriers, which release the active compound in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of anactive compound, as described herein, are prepared by modifyingfunctional groups present in the active compound in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent active compound. Prodrugs include compounds wherein ahydroxy, amino or mercapto group is bonded to any group that, when theprodrug of the active compound is administered to a mammalian subject,cleaves to form a free hydroxy, free amino or free mercapto group,respectively. Examples of prodrugs include, but are not limited to,acetate, formate and benzoate derivatives of alcohol or amine functionalgroups in the active compounds and the like.

The term “modulate” as used herein, means to interact with a targeteither directly or indirectly so as to alter the activity of the target,including, by way of example only, to enhance the activity of thetarget, to inhibit the activity of the target, to limit the activity ofthe target, or to extend the activity of the target.

The term “modulator” as used herein, refers to a molecule that interactswith a target either directly or indirectly. The interactions include,but are not limited to, the interactions of an agonist, partial agonist,an inverse agonist, antagonist, degrader, or combinations thereof. Insome embodiments, a modulator is an antagonist.

The terms “administer,” “administering”, “administration,” and the like,as used herein, refer to the methods that may be used to enable deliveryof compounds or compositions to the desired site of biological action.These methods include, but are not limited to oral routes, intraduodenalroutes, parenteral injection (including intravenous, subcutaneous,intraperitoneal, intramuscular, intravascular or infusion), topical andrectal administration. Those of skill in the art are familiar withadministration techniques that can be employed with the compounds andmethods described herein. In some embodiments, the compounds andcompositions described herein are administered orally. In someembodiments, the compounds and compositions described herein areadministered intravenously.

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient, and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered, which will relieve to some extent one or more of thesymptoms of the disease or condition being treated. The result includesreduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forexample, an “effective amount” for therapeutic uses is the amount of thecomposition comprising a compound as disclosed herein required toprovide a clinically significant decrease in disease symptoms. Anappropriate “effective” amount in any individual case is optionallydetermined using techniques, such as a dose escalation study.

The terms “enhance” or “enhancing,” as used herein, means to increase orprolong either in potency or duration a desired effect. Thus, in regardto enhancing the effect of therapeutic agents, the term “enhancing”refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of another therapeutic agent in a desiredsystem.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. the LpxC inhibitory compound disclosed herein, or anisotopic variant, tautomer, prodrug, pharmaceutically acceptable salt,solvate, or hydrate thereof, and a co-agent, are both administered to apatient simultaneously in the form of a single entity or dosage. Theterm “non-fixed combination” means that the active ingredients, e.g. theLpxC inhibitory compound disclosed herein, or an isotopic variant,tautomer, prodrug, pharmaceutically acceptable salt, solvate, or hydratethereof, and a co-agent, are administered to a patient as separateentities either simultaneously, concurrently or sequentially with nospecific intervening time limits, wherein such administration provideseffective levels of the two compounds in the body of the patient. Thelatter also applies to cocktail therapy, e.g. the administration ofthree or more active ingredients.

The terms “article of manufacture” and “kit” are used as synonyms.

The term “subject” or “patient” encompasses mammals. Examples of mammalsinclude, but are not limited to, any member of the Mammalian class:humans, non-human primates such as chimpanzees, and other apes andmonkey species; farm animals such as cattle, horses, sheep, goats,swine; domestic animals such as rabbits, dogs, and cats; laboratoryanimals including rodents, such as rats, mice and guinea pigs, and thelike. In one aspect, the mammal is a human.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating at least one symptom of a diseaseor condition, preventing additional symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

Further Forms of Compound A

“Pharmaceutically acceptable,” as used herein, refers a material, suchas a carrier or diluent, which does not abrogate the biological activityor properties of the compound, and is relatively nontoxic, i.e., thematerial is administered to an individual without causing undesirablebiological effects or interacting in a deleterious manner with any ofthe components of the composition in which it is contained.

The term “pharmaceutically acceptable salt” refers to a form of atherapeutically active agent that consists of a cationic form of thetherapeutically active agent in combination with a suitable anion, or inalternative embodiments, an anionic form of the therapeutically activeagent in combination with a suitable cation. Handbook of PharmaceuticalSalts: Properties, Selection and Use. International Union of Pure andApplied Chemistry, Wiley-VCH 2002. S. M. Berge, L. D. Bighley, D. C.Monkhouse, J. Pharm. Sci. 1977, 66, 1-19. P. H. Stahl and C. G. Wermuth,editors, Handbook of Pharmaceutical Salts: Properties, Selection andUse, Weinheim/Ziirich:Wiley-VCH/VHCA, 2002. Pharmaceutical saltstypically are more soluble and more rapidly soluble in stomach andintestinal juices than non-ionic species and so are useful in soliddosage forms. Furthermore, because their solubility often is a functionof pH, selective dissolution in one or another part of the digestivetract is possible and this capability can be manipulated as one aspectof delayed and sustained release behaviours. Also, because thesalt-forming molecule can be in equilibrium with a neutral form, passagethrough biological membranes can be adjusted.

In some embodiments, pharmaceutically acceptable salts are obtained byreacting a compound disclosed herein with an acid. In some embodiments,the compound disclosed herein (i.e. free base form) is basic and isreacted with an organic acid or an inorganic acid. Inorganic acidsinclude, but are not limited to, hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid, and metaphosphoric acid.Organic acids include, but are not limited to, 1-hydroxy-2-naphthoicacid; 2,2-dichloroacetic acid; 2-hydroxyethanesulfonic acid;2-oxoglutaric acid; 4-acetamidobenzoic acid; 4-aminosalicylic acid;acetic acid; adipic acid; ascorbic acid (L); aspartic acid (L);benzenesulfonic acid; benzoic acid; camphoric acid (+);camphor-10-sulfonic acid (+); capric acid (decanoic acid); caproic acid(hexanoic acid); caprylic acid (octanoic acid); carbonic acid; cinnamicacid; citric acid; cyclamic acid; dodecylsulfuric acid;ethane-1,2-disulfonic acid; ethanesulfonic acid; formic acid; fumaricacid; galactaric acid; gentisic acid; glucoheptonic acid (D); gluconicacid (D); glucuronic acid (D); glutamic acid; glutaric acid;glycerophosphoric acid; glycolic acid; hippuric acid; isobutyric acid;lactic acid (DL); lactobionic acid; lauric acid; maleic acid; malic acid(−L); malonic acid; mandelic acid (DL); methanesulfonic acid;naphthalene-1,5-disulfonic acid; naphthalene-2-sulfonic acid; nicotinicacid; oleic acid; oxalic acid; palmitic acid; pamoic acid; phosphoricacid; proprionic acid; pyroglutamic acid (−L); salicylic acid; sebacicacid; stearic acid; succinic acid; sulfuric acid; tartaric acid (+L);thiocyanic acid; toluenesulfonic acid (p); and undecylenic acid.

In some embodiments, pharmaceutically acceptable salts are obtained byreacting a compound disclosed herein with a base. In some embodiments,the compound disclosed herein is acidic and is reacted with a base. Insuch situations, an acidic proton of the compound disclosed herein isreplaced by a metal ion, e.g., lithium, sodium, potassium, magnesium,calcium, or an aluminum ion. In some cases, compounds described hereincoordinate with an organic base, such as, but not limited to,ethanolamine, diethanolamine, triethanolamine, tromethamine, meglumine,N-methylglucamine, dicyclohexylamine, tris(hydroxymethyl)methylamine. Inother cases, compounds described herein form salts with amino acids suchas, but not limited to, arginine, lysine, and the like. Acceptableinorganic bases used to form salts with compounds that include an acidicproton, include, but are not limited to, aluminum hydroxide, calciumhydroxide, potassium hydroxide, sodium carbonate, potassium carbonate,sodium hydroxide, lithium hydroxide, and the like. In some embodiments,the compounds provided herein are prepared as a sodium salt, calciumsalt, potassium salt, magnesium salt, meglumine salt, N-methylglucaminesalt or ammonium salt.

It should be understood that a reference to a pharmaceuticallyacceptable salt includes the solvent addition forms. In someembodiments, solvates contain either stoichiometric ornon-stoichiometric amounts of a solvent, and are formed during theprocess of crystallization with pharmaceutically acceptable solventssuch as water, ethanol, and the like. Hydrates are formed when thesolvent is water, or alcoholates are formed when the solvent is alcohol.Solvates of compounds described herein are conveniently prepared orformed during the processes described herein. In addition, the compoundsprovided herein optionally exist in unsolvated as well as solvatedforms.

Therapeutic agents that are administrable to mammals, such as humans,must be prepared by following regulatory guidelines. Such governmentregulated guidelines are referred to as Good Manufacturing Practice(GMP). GMP guidelines outline acceptable contamination levels of activetherapeutic agents, such as, for example, the amount of residual solventin the final product. Preferred solvents are those that are suitable foruse in GMP facilities and consistent with industrial safety concerns.Categories of solvents are defined in, for example, the InternationalConference on Harmonization of Technical Requirements for Registrationof Pharmaceuticals for Human Use (ICH), “Impurities: Guidelines forResidual Solvents, Q3C(R3), (November 2005).

Solvents are categorized into three classes. Class 1 solvents are toxicand are to be avoided. Class 2 solvents are solvents to be limited inuse during the manufacture of the therapeutic agent. Class 3 solventsare solvents with low toxic potential and of lower risk to human health.Data for Class 3 solvents indicate that they are less toxic in acute orshort-term studies and negative in genotoxicity studies.

Class 1 solvents, which are to be avoided, include: benzene; carbontetrachloride; 1,2-dichloroethane; 1,1-dichloroethene; and1,1,1-trichloroethane.

Examples of Class 2 solvents are: acetonitrile, chlorobenzene,chloroform, cyclohexane, 1,2-dichloroethene, dichloromethane,1,2-dimethoxyethane, N,N-dimethylacetamide, N,N-dimethylformamide,1,4-dioxane, 2-ethoxyethanol, ethyleneglycol, formamide, hexane,methanol, 2-methoxyethanol, methylbutyl ketone, methylcyclohexane,N-methylpyrrolidine, nitromethane, pyridine, sulfolane, tetralin,toluene, 1,1,2-trichloroethene and xylene.

Class 3 solvents, which possess low toxicity, include: acetic acid,acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butylmethylether (MTBE), cumene, dimethyl sulfoxide, ethanol, ethyl acetate, ethylether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropylacetate, methyl acetate, 3-methyl-1-butanol, methylethyl ketone,methylisobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol,1-propanol, 2-propanol, propyl acetate, and tetrahydrofuran.

Residual solvents in active pharmaceutical ingredients (APIs) originatefrom the manufacture of API. In some cases, the solvents are notcompletely removed by practical manufacturing techniques. Appropriateselection of the solvent for the synthesis of APIs may enhance theyield, or determine characteristics such as crystal form, purity, andsolubility. Therefore, the solvent is a critical parameter in thesynthetic process.

In some embodiments, compositions comprising Compound A, comprise anorganic solvent(s). In some embodiments, compositions comprisingCompound A include a residual amount of an organic solvent(s). In someembodiments, compositions comprising Compound A comprise a residualamount of a Class 3 solvent. In some embodiments, the Class 3 solvent isselected from the group consisting of acetic acid, acetone, anisole,1-butanol, 2-butanol, butyl acetate, tert-butylmethyl ether, cumene,dimethyl sulfoxide, ethanol, ethyl acetate, ethyl ether, ethyl formate,formic acid, heptane, isobutyl acetate, isopropyl acetate, methylacetate, 3-methyl-1-butanol, methylethyl ketone, methylisobutyl ketone,2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, propylacetate, and tetrahydrofuran. In some embodiments, the Class 3 solventis selected from ethyl acetate, isopropyl acetate,tert-butylmethylether, heptane, isopropanol, and ethanol.

In some embodiments, the compositions comprising Compound A include adetectable amount of an organic solvent. In some embodiments, theorganic solvent is a Class 3 solvent.

In other embodiments are compositions comprising Compound A wherein thecomposition comprises a detectable amount of solvent that is less thanabout 1%, wherein the solvent is selected from acetone,1,2-dimethoxyethane, acetonitrile, ethyl acetate, tetrahydrofuran,methanol, ethanol, heptane, and 2-propanol. In a further embodiment arecompositions comprising Compound A wherein the composition comprises adetectable amount of solvent which is less than about 5000 ppm. In yet afurther embodiment are compositions comprising Compound A, wherein thedetectable amount of solvent is less than about 5000 ppm, less thanabout 4000 ppm, less than about 3000 ppm, less than about 2000 ppm, lessthan about 1000 ppm, less than about 500 ppm, or less than about 100ppm.

The methods and formulations described herein include the use ofN-oxides (if appropriate), or pharmaceutically acceptable salts ofcompounds having the structure disclosed herein, as well as activemetabolites of these compounds having the same type of activity.

In some embodiments, sites on the organic radicals (e.g. alkyl groups,aromatic rings) of compounds disclosed herein are susceptible to variousmetabolic reactions. Incorporation of appropriate substituents on theorganic radicals will reduce, minimize or eliminate this metabolicpathway. In specific embodiments, the appropriate substituent todecrease or eliminate the susceptibility of the aromatic ring tometabolic reactions is, by way of example only, a halogen, deuterium, analkyl group, a haloalkyl group, or a deuteroalkyl group.

In another embodiment, the compounds described herein are labeledisotopically (e.g. with a radioisotope) or by another other means,including, but not limited to, the use of chromophores or fluorescentmoieties, bioluminescent labels, or chemiluminescent labels.

Compounds described herein include isotopically-labeled compounds, whichare identical to those recited in the various formulae and structurespresented herein, but for the fact that one or more atoms are replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, sulfur, fluorine chlorine, iodine,phosphorus, such as, for example, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S,¹⁸F, ³⁶Cl, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ³²P and ³³P. In one aspect,isotopically-labeled compounds described herein, for example those intowhich radioactive isotopes such as ³H and ¹⁴C are incorporated, areuseful in drug and/or substrate tissue distribution assays. In oneaspect, substitution with isotopes such as deuterium affords certaintherapeutic advantages resulting from greater metabolic stability, suchas, for example, increased in vivo half-life or altered metabolicpathways to reduce undesirable metabolites or reduced dosagerequirements.

In some embodiments, one or more hydrogen atoms on Compound A arereplaced with deuterium. In some embodiments, substitution withdeuterium affords certain therapeutic advantages resulting from greatermetabolic stability, such as, for example, increased in vivo half-lifeor reduced dosage requirements.

In one aspect, described is a compound with the following structure:

-   -   wherein,    -   each R is independently selected from hydrogen or deuterium,    -   or an isotopic variant, tautomer, pharmaceutically acceptable        salt, solvate, or hydrate thereof.

In some embodiments, the compounds disclosed herein possess one or morestereocenters and each stereocenter exists independently in either the Ror S configuration. For example, in some embodiments, the compounddisclosed herein exists in the R configuration when one stereocenter ispresent. In other embodiments, the compound disclosed herein exists inthe S configuration when one stereocenter is present. In someembodiments, the compound disclosed herein exists in the RRconfiguration when two stereocenters are present. In some embodiments,the compound disclosed herein exists in the RS configuration when twostereocenters are present. In some embodiments, the compound disclosedherein exists in the SS configuration when two stereocenters arepresent. In some embodiments, the compound disclosed herein exists inthe SR configuration when two stereocenters are present.

The compounds presented herein include all diastereomeric, individualenantiomers, atropisomers, and epimeric forms as well as the appropriatemixtures thereof. The compounds and methods provided herein include allcis, trans, syn, anti, entgegen (E), and zusammen (Z) isomers as well asthe appropriate mixtures thereof.

Individual stereoisomers are obtained, if desired, by methods such as,stereoselective synthesis and/or the separation of stereoisomers bychiral chromatographic columns or the separation of diastereomers byeither non-chiral or chiral chromatographic columns or crystallizationand recrystallization in a proper solvent or a mixture of solvents. Incertain embodiments, compounds disclosed herein are prepared as theirindividual stereoisomers by reacting a racemic mixture of the compoundwith an optically active resolving agent to form a pair ofdiastereoisomeric compounds/salts, separating the diastereomers andrecovering the optically pure individual enantiomers. In someembodiments, resolution of individual enantiomers of compounds disclosedherein is carried out using covalent diastereomeric derivatives of thecompounds described herein. In another embodiment, diastereomers ofcompounds disclosed herein are separated by separation/resolutiontechniques based upon differences in solubility. In other embodiments,separation of stereoisomers of compounds disclosed herein is performedby chromatography or by the forming diastereomeric salts and separationby recrystallization, or chromatography, or any combination thereof.Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers, Racemates andResolutions”, John Wiley And Sons, Inc., 1981. In some embodiments,stereoisomers are obtained by stereoselective synthesis.

Separation of individual enantiomers from a racemic mixture is possibleby the use of chiral supercritical fluid chromatography (SFC) or chiralhigh performance liquid chromatography (HPLC). In some embodiments,enantiomers described herein are separated from each other by the use ofchiral SFC or chiral HPLC. In some embodiments, compounds disclosedherein that include one or more chiral centers (e.g. compounds disclosedherein that include the moietytrans-octahydro-1H-pyrido[3,4-b]morpholin-6-yl) are separated intoindividual enantiomers using chiral SFC or chiral HPLC. A wide varietyof conditions and suitable columns are available.

Daicel polysaccharide chiral stationary phases (CSPs) are among thecolumns used for chiral SFC separations. In some embodiments, Daicelanalytical immobilised and coated CHIRALPAK and CHIRALCEL HPLC columnscan be used for SFC analysis.

In some embodiments, screening for the suitability of using a SFC columnis performed on the four main immobilised phases (CHIRALPAK IA, IB, ICand ID) and the four main coated columns (CHIRALPAK AD and AS andCHIRALCEL OD and OJ), with varying concentrations of organic modifier. Avariety of column phases are available, including but not limited to ODand OJ, OX and OZ chlorinated phases, and a range of complementarycellulose based CHIRALCEL phases including OA, OB, OC, OF, OG and OK.

Non-limiting examples of chiral selectors contemplated for use in theseparation of enantiomers include amylose tris (3,5-dimethylphenylcarbamate), cellulose tris (3,5-dimethylphenylcarbamate), cellulose tris (3,5-dichlorophenylcarbamate), amylose tris (3-chlorophenylcarbamate),amylose tris (3, 5-dichlorophenylcarbamate), amylose tris (3-chloro,4-methylphenylcarbamate), amylose tris((S)-alpha-methylbenzylcarbamate), amylose tris(5-chloro-2-methylphenylcarbamate), cellulose tris (4-methylbenzoate),cellulose tris (4-chloro-3-methylphenylcarbamate), and cellulose tris(3-chloro-4-methylphenylcarbamate).

Non-limiting examples of chiral columns contemplated for use in theseparation of enantiomers include CHIRALPAK IA SFC, CHIRALPAK AD-H SFC,CHIRALPAK IB SFC, CHIRALCEL OD-H SFC, CHIRALPAK IC SFC, CHIRALPAK IDSFC, CHIRALPAK IE SFC, CHIRALPAK IF SFC, CHIRALPAK AZ-H SFC, CHIRALPAKAS-H SFC, CHIRALPAK AY-H SFC, CHIRALCEL OJ-H SFC, CHIRALCEL OX-H SFC,and CHIRALCEL OZ-H SFC.

In additional or further embodiments, the compounds described herein aremetabolized upon administration to an organism in need to produce ametabolite that is then used to produce a desired effect, including adesired therapeutic effect.

A “metabolite” of a compound disclosed herein is a derivative of thatcompound that is formed when the compound is metabolized. The term“active metabolite” refers to a biologically active derivative of acompound that is formed when the compound is metabolized. The term“metabolized,” as used herein, refers to the sum of the processes(including, but not limited to, hydrolysis reactions and reactionscatalyzed by enzymes) by which a particular substance is changed by anorganism. Thus, enzymes may produce specific structural alterations to acompound. For example, cytochrome P450 catalyzes a variety of oxidativeand reductive reactions while uridine diphosphate glucuronyltransferasescatalyze the transfer of an activated glucuronic-acid molecule toaromatic alcohols, aliphatic alcohols, carboxylic acids, amines and freesulphydryl groups. Metabolites of the compounds disclosed herein areoptionally identified either by administration of compounds to a hostand analysis of tissue samples from the host, or by incubation ofcompounds with hepatic cells in vitro and analysis of the resultingcompounds.

Pharmaceutical Compositions

In certain embodiments, the heterocyclic LpxC inhibitory compound asdescribed herein is administered as a pure chemical. In otherembodiments, the heterocyclic LpxC inhibitory compound described hereinis combined with a pharmaceutically suitable or acceptable carrier (alsoreferred to herein as a pharmaceutically suitable (or acceptable)excipient, physiologically suitable (or acceptable) excipient, orphysiologically suitable (or acceptable) carrier) selected on the basisof a chosen route of administration and standard pharmaceutical practiceas described, for example, in Remington: The Science and Practice ofPharmacy (Gennaro, 21^(st) Ed. Mack Pub. Co., Easton, PA (2005)).

Provided herein is a pharmaceutical composition comprising at least oneheterocyclic LpxC inhibitory compound as described herein, or astereoisomer, pharmaceutically acceptable salt, or N-oxide thereof,together with one or more pharmaceutically acceptable carriers. Thecarrier(s) (or excipient(s)) is acceptable or suitable if the carrier iscompatible with the other ingredients of the composition and notdeleterious to the recipient (i.e., the subject or patient) of thecomposition.

One embodiment provides a pharmaceutical composition comprising CompoundA, or an isotopic variant, tautomer, prodrug, pharmaceuticallyacceptable salt, solvate, or hydrate thereof, and at least onepharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition is in a dosage formfor dosing or administration by injection. In some embodiments, thepharmaceutical composition is in a dosage form for intravenous (I.V.)injection or infusion, or intramuscular, subcutaneous, or intradermalinjection. In some embodiments, the pharmaceutical composition is in adosage form for I.V. injection or infusion. In some embodiments, thepharmaceutical composition is a solution.

In some embodiments, the pharmaceutical composition is in a dosage formfor oral dosing or administration. In some embodiments, the dosage formis a liquid. In some embodiments, the dosage form is a suspension,solution, syrup, or elixir. In some embodiments, the dosage form is asuspension. In some embodiments, the dosage form is a nanosuspension. Insome embodiments, the dosage form is a solution. In other embodiments,the dosage form is a tablet or a capsule.

In some embodiments, the at least one pharmaceutically acceptableexcipient is a co-solvent, oil, surfactant, complexing agent, asolubilizing polymer, a P-gp modulator, a buffering agent, or acombination thereof.

In certain embodiments, the heterocyclic LpxC inhibitory compounddisclosed herein is substantially pure, in that it contains less thanabout 5%, or less than about 1%, or less than about 0.1%, of otherorganic small molecules, such as unreacted intermediates or synthesisby-products that are created, for example, in one or more of the stepsof a synthesis method.

Pharmaceutical compositions are administered in a manner appropriate tothe disease to be treated (or prevented). An appropriate dose and asuitable duration and frequency of administration will be determined bysuch factors as the condition of the patient, the type and severity ofthe patient's disease, the particular form of the active ingredient, andthe method of administration. In general, an appropriate dose andtreatment regimen provides the composition(s) in an amount sufficient toprovide therapeutic and/or prophylactic benefit (e.g., an improvedclinical outcome), or a lessening of symptom severity. Optimal doses aregenerally determined using experimental models and/or clinical trials.The optimal dose depends upon the body mass, weight, or blood volume ofthe patient.

LpxC, Lipid A and Gram-Negative Bacteria

Metalloproteins influence a vast diversity of biological systems,biological processes, and diseases. For example,UDP-{3—O—[(R)-3-hydroxymyristoyl]})-N-acetylglucosamine deacetylase(LpxC) is an essential enzyme involved in the first committed step inlipid A biosynthesis for gram-negative bacteria. Lipid A is an essentialcomponent of the outer membrane of gram-negative bacteria. LpxC is azinc(II)-dependent metalloenzyme, with two histidines and an asparticacid residue bound to the zinc(II) ion. Structures of LpxC show thezinc(II) ion is bound to two water molecules, both of which have beenimplicated in the mechanism of the enzyme. LpxC is highly conservedacross strains of gram-negative bacteria, making LpxC an attractivetarget to treat gram-negative infections.

In recent years, there has been an increase in resistant and multi-drugresistant strains of bacteria. Thus, there is a need for newantibiotics, especially with new mechanisms of action. There remains aneed for metalloprotein modulators of LpxC useful in the field oftherapeutics, diagnostics, and research.

One embodiment provides a method of inhibitingUDP-{3—O—[(R)-3-hydroxymyristoyl]}-N-acetylglucosamine deacetylaseenzyme comprising contacting the enzyme with the LpxC inhibitorycompound disclosed herein.

Methods of Treatment

Disclosed herein are methods of treating disease wherein the inhibitionof bacterial growth is indicated. Such disease includes gram-negativebacterial infection. In some embodiments, the method of treating agram-negative bacterial infection in a patient in need thereof comprisesadministering to the patient a pharmaceutical composition comprising theLpxC inhibitory compound disclosed herein, or an isotopic variant,tautomer, prodrug, pharmaceutically acceptable salt, solvate, or hydratethereof, and a pharmaceutically acceptable excipient. In someembodiments, the gram-negative bacterial infection is selected frompneumonia, sepsis, cystic fibrosis, intra-abdominal infection, skininfections and urinary tract infection. In some embodiments, thegram-negative bacterial infection is a urinary tract infection (UTI), ahospital acquired/ventilator-associated pneumonia (HAP/VAP), or anintra-abdominal infection (IAI). In some embodiments, the gram-negativebacterial infection is selected from chronic urinary tract infections,complicated urinary tract infections, cystitis, pyelonephritis,urethritis, recurrent urinary tract infections, bladder infections,urethral infections, and kidney infections. In some embodiments, thecompounds described herein are used for the treatment of chronic urinarytract infections. In some embodiments, the compounds described hereinare used for the treatment of complicated urinary tract infections. Inother embodiments, the compounds described herein are used for thetreatment of complicated intra-abdominal infection. In some embodiments,the compounds described herein are used for the treatment of chronicintra-abdominal infection. In other embodiments, the compounds describedherein are used for the treatment of hospital acquired pneumonia (HAP)or ventilator associated pneumonia (VAP). In some embodiments theadministration is to treat an existing infection. In some embodimentsthe administration is provided as prophylaxis.

In some embodiments, the LpxC inhibitory compound described herein, oran isotopic variant, tautomer, prodrug, pharmaceutically acceptablesalt, solvate, or hydrate thereof, is used for treating conditionscaused by the bacterial production of endotoxin and, in particular, bygram-negative bacteria and bacteria that use LpxC in the biosynthesis oflipopolysaccharide (LPS) or endotoxin. In some embodiments, the methodof treating a condition caused by endotoxin or LPS in a patient in needthereof comprises administering to the patient a pharmaceuticalcomposition comprising the LpxC inhibitory compound disclosed herein, oran isotopic variant, tautomer, prodrug, pharmaceutically acceptablesalt, solvate, or hydrate thereof, and a pharmaceutically acceptableexcipient. In another embodiment, the heterocyclic LpxC inhibitorycompound and formulations as described herein are useful in thetreatment of conditions that are caused or exacerbated by the bacterialproduction of lipid A and LPS or endotoxin, such as sepsis, septicshock, systemic inflammation, localized inflammation, chronicobstructive pulmonary disease (COPD) and acute exacerbations of chronicbronchitis (AECB). In some embodiments, the method of treating acondition caused by endotoxin or LPS in a patient in need thereofcomprises administering to the patient a pharmaceutical compositioncomprising the LpxC inhibitory compound disclosed herein, or an isotopicvariant, tautomer, prodrug, pharmaceutically acceptable salt, solvate,or hydrate thereof, and a pharmaceutically acceptable excipient, whereinthe condition caused by endotoxin or LPS is selected from sepsis, septicshock, systemic inflammation, localized inflammation, chronicobstructive pulmonary disease (COPD) and acute exacerbations of chronicbronchitis (AECB).

In other embodiments, the LpxC inhibitory compound described herein, oran isotopic variant, tautomer, prodrug, pharmaceutically acceptablesalt, solvate, or hydrate thereof, can be used for the treatment of aserious or chronic respiratory tract infection or complicated urinarytract infections including serious lung and nosocomial infections suchas those caused by Eniterobacter aerogenes, Enterobacter cloacae,Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Kiyveraascorbata, Kluyvera cryocrescense, Shigella sonnei. Proteus mirabilis,Serratia marcesceins, Stenotrophomonas maltophilia, Pseudomonasaeruginosa, Burkholderia cepacia, Acinetobacter baumannii, Alcaligenesxylosoxidans. Flavobacterium meningosepticum, Providencia shiarlii andCirobacter freundii, Haemophilus influenzae, Kluyvera species,Legionella species, Moraxella catarrhalis, Enterobacter species,Acinetobacter species, Klebsiella species, Burkholderia species andProteus species, and infections caused by other bacterial species suchas Neisseria species, Shigella species, Salmonella species, Helicobacterpylori, Vibrionaceae and Bordelella species as well as the infectionscaused by a Brucella species, Francisella tularensis and/or Yersiniapestis.

In one embodiment provided herein is a method of treating agram-negative bacterial infection in a patient in need thereofcomprising administering to the patient a pharmaceutical compositioncomprising the LpxC inhibitory compound disclosed herein, or an isotopicvariant, tautomer, prodrug, pharmaceutically acceptable salt, solvate,or hydrate thereof, and at least one pharmaceutically acceptableexcipient.

One embodiment provides a method wherein the gram-negative bacterialinfection is selected from pneumonia, sepsis, cystic fibrosis,intra-abdominal infection, skin infection and urinary tract infection.

One embodiment provides a method wherein the gram-negative bacterialinfection is selected from chronic urinary tract infection, complicatedurinary tract infection, cystitis, pyelonephritis, urethritis, recurrenturinary tract infections, bladder infections, urethral infections andkidney infections.

One embodiment provides a method wherein the gram-negative bacterialinfection is chronic urinary tract infections. One embodiment provides amethod wherein the gram-negative bacterial infection is complicatedurinary tract infections. One embodiment provides a method wherein theadministration is to treat an existing infection. One embodimentprovides a method wherein the administration is provided as prophylaxis.

In some embodiments, the LpxC inhibitory compound described herein, oran isotopic variant, tautomer, prodrug, pharmaceutically acceptablesalt, solvate, or hydrate thereof, is not active against gram-positivebacteria. In some embodiments, the LpxC inhibitory compound describedherein, or an isotopic variant, tautomer, prodrug, pharmaceuticallyacceptable salt, solvate, or hydrate thereof, is not active againstStaphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes,Bacillus thuringiensis, Lactobacillus rhamnosus, Staphylococcusepidermidis, Bifidobacterium breve, Clostridium difficile, Clostridiumsordelli, Peptostreptococcus anaerobius, Streptococcus pneumoniae,Corynebacterium jeikeium, Propionibacterium acnes, Listeriamonocytogenes, and/or Nocardia cyriacigeorgica complex.

Most gut bacteria are Gram-positive, including C. dificile. Therefore,in some embodiments, the lack of activity against gram-positive bacteriais a benefit. In some embodiments, use of the LpxC inhibitory compounddescribed herein, or an isotopic variant, tautomer, prodrug,pharmaceutically acceptable salt, solvate, or hydrate thereof, to treata gram-negative bacterial infection, as described herein, has no effecton the gut microflora and thus reduces the risk of secondary infectionsfrom, for example, C. difficile.

Combination Therapy

In some instances, Gram-negative bacteria are more resistant to a largernumber of antibacterials and chemotherapeutic agents than aregram-positive bacteria due in part to their outer membrane, which actsas an efficient permeability barrier.

A survey of recently reported antibacterials of natural origin showedthat over 90% lacked activity against Escherichia coli, although theywere active against gram-positive bacteria. Young and Silver (J.Bacteriol. 173(12):3609-14 (1991)) demonstrated that an envA1 strain,having an altered outer membrane, is sensitive to a variety of large andhydrophobic antibacterials to which wild type E. coli is resistant.Additionally, Vaara, et al., (Antimicrobial Agents and Chemotherapy37(11):2255-2260 (1993)) review a variety of outer membrane-defectivemutants of E. coli and S. typhimurium that show greater susceptibilitythan the corresponding wild type strain to a variety of antibacterialagents.

In some embodiments, the present invention provides synergisticcombinations of antibacterial agents with the LpxC inhibitory compoundor pharmaceutical compositions disclosed herein. In some embodiments,the LpxC inhibitory compound disclosed herein has both intrinsicantibacterial properties as well the ability to improve permeability ofthe outer membrane of gram-negative bacteria to other antibacterialagents. In some embodiments, the antibacterial agent is selected fromthe group consisting of vancomycin, linezolid, azithromycin, imipenem,teicoplanin, daptomycin, clindamycin, rifampin, cefotaxime, gentamicin,novobiocin, and telavancin.

The use of such synergistic combinations of drugs could have manyadvantages over conventional single compound therapy, including loweredside-effects of the antibacterial agent due to lower doses used or toshorter time of treatment, more rapid cure of infection shorteninghospital stays, increasing spectrum of pathogens controlled, anddecreasing incidence of development of resistance to antibiotics.

Articles of Manufacture and Kits

Disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more methods described herein. In someembodiments, additional components of the kit comprises a carrier,package, or container that is compartmentalized to receive one or morecontainers such as vials, tubes, and the like, each of the container(s)comprising one of the separate elements to be used in a method describedherein. Suitable containers include, for example, bottles, vials,plates, syringes, and test tubes. In one embodiment, the containers areformed from a variety of materials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, bottles, tubes, bags, containers, and any packaging materialsuitable for a selected formulation and intended mode of use.

For example, the container(s) include one or more of the compoundsdescribed herein. Such kits optionally include an identifyingdescription or label or instructions relating to its use in the methodsdescribed herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

Other embodiments and uses will be apparent to one skilled in the art inlight of the present disclosures. The following examples are providedmerely as illustrative of various embodiments and shall not be construedto limit the invention in any way.

EXAMPLES I. Chemical Synthesis

Unless otherwise noted, reagents and solvents were used as received fromcommercial suppliers. Anhydrous solvents and oven-dried glassware wereused for synthetic transformations sensitive to moisture and/or oxygen.Yields were not optimized. Reaction times are approximate and were notoptimized. Column chromatography and thin layer chromatography (TLC)were performed on silica gel unless otherwise noted. Spectra are givenin ppm (δ) and coupling constants, J are reported in Hertz. For protonspectra the solvent peak was used as the reference peak.

The following abbreviations and terms have the indicated meaningsthroughout:

-   -   AcOH or HOAc=acetic acid    -   Amb=ambient temperature and humidity    -   ATCC=American Type Culture Collection    -   AUC=area under the curve    -   Bn=benzyl    -   BnOH=benzyl alcohol    -   CPME=cyclopropyl methyl ether    -   CV=coefficient of variation    -   DBU=1,8-diazabicyclo[5.4.0]undec-7-ene    -   DCE=1,2-dichloroethane    -   DCM=dichloromethane or methylene chloride    -   DMF=N,N-dimethylformamide    -   DSM=German Collection of Microorganisms    -   eq or eq. or equiv.=equivalent(s)    -   Et=ethyl    -   Et₃N=triethylamine    -   EtOAc or EA=ethyl acetate    -   EtOH=ethanol    -   g=gram    -   h or hr=hour    -   HPβCD=(2-hydroxypropyl)-o-cyclodextrin    -   HP-55=hydroxypropyl methylcellulose phthalate    -   HPLC=high pressure liquid chromatography    -   HPMC 606=hydroxypropyl methylcellulose 606    -   IPA=isopropyl alcohol    -   kg or Kg=kilogram    -   L=liter    -   LC/MS=LCMS=liquid chromatography-mass spectrometry    -   LiHMDS=lithium bis(trimethylsilyl)amide or lithium        hexamethyldisilazide    -   LpxC=UDP-{3—O—[(R)-3-hydroxymyristoyl]}-N-acetylglucosamine        deacetylase    -   LRMS=low resolution mass spectrometry    -   m/z=mass-to-charge ratio    -   mCPBA=meta-chloroperoxybenzoic acid    -   Me=methyl    -   MeOH=methanol    -   mg=milligram    -   MIC=minimum inhibitory concentration    -   min=minute    -   mL=milliliter    -   mmol=millimole    -   MTBE=methyl tert-butyl ether    -   NaBH(OAc)₃=sodium triacetoxyborohydride    -   NaOEt=sodium ethoxide    -   NCTC=National Collection of Type Cultures    -   NEQAS=United Kingdom National External Quality Assessment Scheme    -   NMM=N-methylmorpholine    -   NMR=nuclear magnetic resonance    -   PBS=phosphate buffered saline    -   PdCl₂(PPh₃)₂=dichiorobis(triphenylphosphine)palladium(II)    -   Ph=phenyl    -   rt or RT=room temperature    -   SBEPCD=sulfobutylether-o-cyclodextrin    -   SDD=spray-dried dispersion    -   sec-Bu-NH₂=sec-butyl amine    -   TEMPO=(2,2,6,6-Tetramethylpiperidin-1-yl)oxyl    -   TFA=trifluoroacetic acid    -   TFAA=trifluoroacetic anhydride    -   THF=tetrahydrofuran    -   vitamin E TPGS=D-α-tocopheryl polyethylene glycol 1000 succinate    -   Vol. or vol.=volume or volumes    -   XRPD=X-ray powder diffraction

In some instances, Compound A is synthesized as outlined in Schemes A-D.

Example 1: Preparation of(S)-1-(3-(5,6-dihydroxypyrimidin-4-yl)-2-(4-((4-(morpholinomethyl)phenyl)ethynyl)phenyl)propyl)azetidine-3-carbonitrile(Compound A)

Example 1-1: Preparation of ethyl 2-(benzyloxy)acetate (2A)

To a 3-L, three-necked round-bottom flask was charged2-(benzyloxy)acetic acid (1A, 497.6 g, 3.00 mol, 1.0 eq.), followed byEtOH (1.65 mL, 3.3 vol.) at ambient temperature. To the stirred reactionmixture was added concentrated sulfuric acid (4.20 mL) dropwise over 2min. After addition the mixture was heated to reflux for 23 h. Aftercooling to 40-50° C. the mixture was concentrated under vacuum. Theresulting light yellow liquid was diluted with EtOAc (1.50 L, 3.0 vol.),and washed with saturated aqueous K₂HPO₄ (250 mL×2) and brine (250 mL).The organic phase was concentrated under vacuum to provide compound 2Aas light yellow liquid (555.5 g, 95.5% yield). ¹H NMR is consistent withthe structure.

Example 1-2: Preparation of ethyl5-(benzyloxy)-6-hydroxypyrimidine-4-carboxylate (4A)

A 1-L, three-necked round-bottom flask was charged with NaH (43.7 g, 60%in mineral oil, 1.09 mol, 1.50 eq.), and then the flask was cooled in anice-water bath and THF (566 mL, 4.0 vol.) was added. After thesuspension was cooled to 0-10° C., 2A (141.5 g, 0.728 mol, 1.0 eq.) wasadded dropwise over 5 min. The addition funnel was washed with THF (71mL) and the rinse was added to the batch over 5 min. Then to the batchwas added diethyl oxalate (143.7 g, 0.983 mol, 1.35 eq.) dropwise over 5min. After addition the suspension was slowly warmed to room temperatureover 1 h and then stirred over 4 days at room temperature. The resultingmixture containing 3A was light brown clear solution.

The reaction mixture was then cooled to 0-10° C. and formamidine acetatesolid (189.6 g, 1.82 mol, 2.5 eq.) was added portion wise over 5 min,followed by the addition of 21% NaOEt solution in EtOH (354 g, 1.09 mol,1.5 eq.) over 5 min, and the reaction mixture was warmed to roomtemperature over 3 h. After stirring overnight at room temperature thereaction mixture was cooled to 0-10° C., then poured into a containerwith pre-cooled 2N HCl (1700 mL, 0-10° C.) and DI-water (420 mL) at0-10° C. over 20 min. The resulting mixture was stirred for another 1 hand then filtered. The cake was washed with DI water (420 mL×2),followed by heptanes (420 mL 2), and then dried at 40-50° C. under highvacuum to provide 4A as a light yellow solid (44% yield over two steps).¹H NMR is consistent with the structure.

Example 1-3: Preparation of ethyl5-(benzyloxy)-6-hydroxypyrimidine-4-carboxylate (5A)

A suspension of 4A (106.0 g, 386.5 mmol, 1.0 eq.) in toluene (954 mL,9.0 vol.) was cooled to 0-10° C. by ice-water bath. To the cooledreaction mixture was added Et₃N (43.0 g, 425.1 mmol, 1.1 eq.), followedby addition of POCl₃ (252.2 mL, 27.1 mol, 7.0 eq.) dropwise over 40 minwhile maintaining the temperature at 0-10° C. After the addition wascomplete the reaction mixture was heated to 85-95° C. and then kept atthis temperature for 1.5 h. The resulting dark solution was concentratedunder vacuum. The resulting dark liquid was diluted with EtOAc (2.0 L)and cooled to 0-10° C. and the pH of the solution was adjusted to >7 bythe addition of saturated aqueous NaHCO₃ (300 mL). After warming to roomtemperature, the organic phase was separated, and filtered through ashort pad of Na₂SO₄ (106 g). The solid cake was washed with EtOAc (200mL) and filtered. The combined filtrate was concentrated to provide 5Aas light dark liquid (111.4 g, 98% yield). HPLC purity: 94.0% (AUC) at254 nm. ¹H NMR is consistent with the structure. This product was usedfor next reaction without further purification.

Example 1-4: Preparation of ethyl5,6-bis(benzyloxy)pyrimidine-4-carboxylate (6A) & benzyl5,6-bis(benzyloxy)pyrimidine-4-carboxylate (7A)

To a solution of 5A (117.6 g, 3865 mmol, 1.0 eq.) in acetonitrile (941mL, 8.0 vol.) at room temperature was added DBU (134.6 g, 2.20 eq),followed by benzyl alcohol (86.9 g, 2.0 eq) dropwise over 20 min. A mildexothermic was observed during benzyl alcohol addition. After theaddition, the clear solution was stirred overnight at room temperatureand then concentrated under vacuum. The resulting residue was dilutedwith EtOAc (1.76 L, 15 vol), washed with water (706 mL, 6.0 vol), 1 NHCl (470 mL, 4.0 vol), and 20% brine (470 mL, 4.0 vol). The organicphase was separated and concentrated to provide a mixture of 6A & 7A asa dark solution (176.1 g, >99%). ¹H NMR is consistent with the presenceof both structures. This product was used for next reaction withoutfurther purification.

Example 1-5: Preparation of (5,6-bis(benzyloxy)pyrimidin-4-yl)methanol(8A)

The mixture of 6A & 7A (140.0 g, 384 mmol, 1.0 eq.) is dissolved in amixed solvent of IPA/MeOH (3/1, 840 mL/280 mL, 8.0 vol.). The reactionmixture was cooled to 0-10° C. and NaBH₄ (9.69 g, 256 mmol, 0.67 equiv.)was added in one portion. The mixture was stirred for 1 h. Thenadditional NaBH₄ (4.85 g, 0.34 eq) was added every 1 h. After total 2.0eq. of NaBH₄ (29.07 g, 768 mmol) was added, the reaction was complete.The batch was cooled back to 0-10° C., saturated aqueous solution ofNH₄Cl (164.4 g, 3.07 mol) in water (1680 mL) over 1.5 h. The mixture waswarmed to room temperature and stirred overnight. After that the slurrywas filtered; the resulting solid cake was slurry-washed with water (420mL×2) to provide first crop of 8A as a white solid. The combinedfiltrated was stirred for 1 h and then filtered to provide second cropof 8A as a yellow solid. The total yield was 66%. ¹H NMR is consistentwith the structure.

Example 1-6: Preparation of 4,5-bis(benzyloxy)-6-(bromomethyl)pyrimidine(9A)

To a stirred solution of 8A (1000 g) in DMF (5.0 vol) cooled to 10° C.using ice bath, was added pyridine (1.2 eq) over a period of 10 minutes.To this PBr₃ (0.80 eq) was added slowly over a period of 30 minutes. Thereaction mixture was stirred at 0° C. over a period of 1 hr and thenquenched with water (8 vol) in an ice bath. The reaction mixture wasextracted with dichloromethane (4.0 vol) and the organic layer was driedover Na₂SO₄ and then filtered. The organic layer was concentrated, thenthe resulting residue was slurried in heptanes and filtered to give a 9A(1.5 kg) as a white solid. ¹H NMR is consistent with the structure.

Example 1-7: Preparation of(S)-4-benzyl-3-(2-(4-iodophenyl)acetyl)oxazolidin-2-one (12A)

To a cloudy solution of (S)-benzyl-2-oxazolidinone (10A, 290.1 g, 1.3eq) and NMM (1.65 L, 1.3 eq) in toluene (1.65 L, 5 vol) was addedpivaloyl chloride (169.6 mL, 1.1 eq) at 19.5-20.2° C. over 9 min. Thecloudy solution was heated to 80° C. and a solution of4-iodophenylacetic acid (11A, 330.0 g, 1.0 eq) and NMM (235.4 mL, 1.7eq) in toluene (660 m L, 2 vol) was added over 135 min. The mixture wasthen heated at 107-112° C. for 5 h. The mixture was cooled to 90-95° C.,and another portion of pivaloyl chloride (66.0 mL, 0.43 eq) was addedand the mixture was heated at 107-112° C. for another 14 h. The mixturewas cooled to 90-95° C., and another portion of pivaloyl chloride (46.2mL, 0.30 eq) was added and the mixture was heated at 107-112° C. foranother 7 h. The mixture was cooled to 90-95° C., and another portion ofpivaloyl chloride (18.5 mL, 0.12 eq) was added and the mixture washeated at 107-112° C. for another 17 h, and at this point the reactionwas deemed to be complete. The reaction was cooled to <35° C. and 7%NaHCO₃ in H₂O (1.32 L) was added (endothermic). After being stirred for20 min, two layers were separated. The organic phase was washed with 7%NaHCO₃ in H₂O (1.32 L), H₂O (1.32 L), and 13% NaCl in H₂O (1.32 L). Theorganic layer (2810 mL) was concentrated to 2.5 vol (˜824 mL) containingtoluene (˜330 mL, 1 vol). The solution was held at it overnight and somecrude product precipitated. The mixture was heated at 35-40° C. todissolve most solids. IPA (1980 mL, 6 vol) was added over 30 min. Theresulting slurry was stirred at 35-40° C. for 2 h, and IPA (1980 mL, 6vol) was added over 20 min at 35-40° C. The slurry was stirred at 35-40°C. for 3 h and at rt for 44 h. The slurry was then gradually cooled to0-5° C. and stirred for 3 h. The solids were filtered, washed with IPA(660 mL/2), dried under high vacuum at 45° C. to provide 12A (405.2 g,76% yield, 98.5% (AUC)) as an off-white solid. ¹H NMR is consistent withthe structure.

Example 1-8: Preparation of(S)-4-benzyl-3-((S)-3-(5,6-bis(benzyloxy)pyrimidin-4-yl)-2-(4-iodophenyl)propanoyl)oxazolidin-2-one(13A)

To a solution of 12A (344.4 g) and LiI (10.4 g, 0.1 eq) in THF (1200 mL,4 vol) was added 1.0 M LiHMDS solution in THF (934.4 mL, 1.2 eq) at−24.8° C. to −18.8° C. over 35 min. After 15 min, 9A (300.0 g, 1.0 eq)in THF (375 mL, 1.25 vol) was added at −24.1° C. to −19.6° C. over 23min. The flask and addition funnel were rinsed with THF (75 mL, 0.25vol). After the addition was complete, the resulting solution wasstirred at −22.8° C. to −17.3° C. for 4 h, at this point the reactionwas deemed to be complete by HPLC analysis. The reaction was quenchedwith 15% NH₄Cl solution in H₂O (900 mL) over 10 min at <20° C. Themixture was warmed to 10.5° C., and MTBE (900 mL, 3 vol) was added. Thetwo phases were separated. The organic layer was washed with 1 M HCl(900 mL, 3 vol), H₂O (900 mL×3). The organic layer was concentratedunder vacuum at 40-50° C. to 3 vol (˜900 mL) and was azeotroped withEtOAc. EtOAc (1200 mL, 4 vol) was added and the mixture was concentratedto 3 vol (900 mL) three times. To the concentrated residue was addedheptanes dropwise (1800 mL, 6.0 vol mL) at 60° C. After the addition wascomplete, the solution was stirred at 60° C. for 2 h and then theresulting slurry was cooled to 10° C. over 4 h and stirred at 10° C. for8 h, filtered, washed with EtOAc/heptanes (1:3, 600 mL×2), and driedunder high vacuum at 30-40° C. for 24 h to provide 13A (399.1 g, 70.6%,99.1% AUC, 99.5% de) as a light yellow solid. H NMR is consistent withthe structure.

Example 1-9: Preparation of(S)-3-(5,6-bis(benzyloxy)pyrimidin-4-yl)-2-(4-iodophenyl)propan-1-ol(14A)

To a solution 13A (300.0 g, 413.35 mmol) in THF (750.0 mL, 2.5 vol) andEtOH (1350 mL, 4.5 vol) was added 2 M LiBH₄ in THF (206.67 mL, 1.0equiv) dropwise over 35 min at 20 t 5° C. (T: 17.3-23.0° C.). (Themixture turned to a thick slurry when 80 mL of LiBH₄ was added andturned to a solution again after the completion of the addition.) Afterthe addition was complete, the solution was stirred at 20±5° C. (T:18.8-22.4° C.) for 5 h (97.3% conversion, e.r: 99.3:0.7). 15% NH₄Clsolution in H₂O (900 mL, 3 vol) was added dropwise over 15 min at 15-30°C. MTBE (1200 mL, 4 vol) was added follows by 26% NaCl (300 mL, 1 vol).Two phases were separated, and the organic was washed with 13% NaClsolution in H₂O (900 mL×4). The organics (2.45 L, ¹H NMR, THF, 28.2%;EtOH, 24.5%; MTBE, 47.3% v/v) was concentrated to 780 mL (2.6 vol). IPA(1200 mL, 4 vol) was added and the solution was concentrated to 780 mL(2.6 vol) again. IPA (1200 mL, 4 vol) was added and the solution wasconcentrated to 780 mL (2.6 vol). IPA (1200 mL, 4 vol) was added and thesolution was concentrated to 960 mL (3.2 vol) (¹H NMR, IPA: 96.8%, EtOH:3.2% v/v). H₂O (375 mL, 1.25 vol) was added at 50° C. The batch turnedto a suspension after the addition of H₂O. The precipitation was startedafter 20 min and turned to a thick slurry. The slurry was stirred at 50°C. for 5 h, then cooled to 5° C. over 4 h, and stirred at 5° C. for 12 h(conc. 8.10 mg/mL), filtered, washed with IPA/H₂O (2:1, 300 mL×2), driedunder high vacuum at 35-40° C. for 24 h to afford 14A (202.6 g, 88.7%,98.4% AUC, 99.9% ee) as an off-white solid.

Example 1-10: Preparation of(S)-3-(5,6-bis(benzyloxy)pyrimidin-4-yl)-2-(4-iodophenyl)propanal (15A)

To a solution of 14A (1.5 kg, 2.72 mol, 1.0 equiv) in DCM (12 L, 8 vol,Fisher Chemicals lot #198170) in a 30-L reactor were added TEMPO (4.2 g,0.027 mol, 0.01 equiv, KBr (32.3 g, 0.27 mol, 0.1 equiv), and water(2.25 L, 1.5 vol). The mixture was cooled to 5±5° C., and 10% sodiumhypochlorite (2.3 L, 5.43 mol, 1.1 equiv, pH was adjusted to 9-9.3 with0.59 L of 8% NaHCO₃ solution) was charged to the mixture slowly over aperiod of 1 h while keeping the temperature at 5±5° C. After theaddition was complete, the biphasic mixture was stirred at 5±5° C. foradditional 10 min. The reaction was monitored by HPLC for completion(spec: <4% 14A). Upon completion, the batch was quenched by the slowaddition of 12% Na₂S₂O₃ solution (3 L, 2 vol) over 30 min keeping thebatch temperature 5±5° C. (end point: negative KF-starch paper test).MeOH (3.0 L, 2 vol) was charged to the batch over 1 h with mixing andlet the layers separate. The cloudy organic layer containing crude 15Awas separated and directly used in stage 2, reductive amination.Analysis of 15A: Chemical purity: 93.6% AUC by HPLC.

Example 1-11: Preparation of(S)-1-(3-(5,6-bis(benzyloxy)pyrimidin-4-yl)-2-(4-iodophenyl)propyl)azetidine-3-carbonitrile(17A)

Azetidine-3-carbonitrile (16A) (0.32 kg, 2.73 mol, 1.0 equiv) wascharged into the reactor under nitrogen atmosphere. To this the cloudyorganic phase containing 15A (1.5 kg, 2.72 mol, 1.0 equiv) in DCM (≈12L) and MeOH (3 L, 2 vol) was charged followed by the addition ofadditional MeOH (7.5 L, 6 vol) to form a clear yellow solution. At thispoint, the batch was analyzed by ¹H NMR for the DCM to MeOH ratio(53.7:46.3) and KF (3.98%) for the water content. The batch was cooledto 10 t 5° C. and was added a solution of 2-methyl-pyridine boranecomplex (0.29 kg, 2.73 mol, 1.0 equiv) in THF (0.75 L, 0.5 vol) over 1 hmaintaining the batch temperature 10±5° C. Upon complete charge ofborane complex, the batch was agitated for additional 10 min at 10±5°C., warmed to 20±5° C. over 1 h, and agitated at the temperature untilcompletion (12 h) confirmed by HPLC (spec: >98%). After completion ofreaction, 5% NaHCO₃ (4.5 L, 3 vol,) was added over 30 min and agitatedfor additional 1 h at 10±5° C. Two phases were separated and the aqueousphase was removed. The combined organic phase was washed with 10%aqueous citric acid (3 vol×2, 9 L), 8% NaHCO₃ (4.5 L, 3 vol), and 13%aqueous brine (4.5 L, 3 vol). The organic phase containing 17A wasconcentrated to 3 L (2 vol) under reduced pressure. Then the batch wassubjected to solvent swap by MTBE (3 vol×2, 15 L) to achieve the finalconcentration of DCM to <5 vol % by ¹H NMR (2.5 vol %). The batch volumewas adjusted by MTBE (16.5 L, 11 vol) and agitated at 20±5° C. for atleast 5 h. The insoluble solid separated was removed by filtration andthe filtrate was adjusted to 15 vol by the addition of MTBE (6 L, 4vol). The batch was cooled to 5±5° C. and 3 M HCl solution in CPME (2.27L, 2.5 equiv) was added over 1 h by maintaining the batch at 5±5° C. Theslurry was stirred at 5±5° C. for at least 1 h and filtered. 17A-HCl waswashed with MTBE (2 vol×2, 6 L) and conditioned for additional 4-5 hunder inert atmosphere to remove most of MTBE. The filter cake wasanalyzed for both chemical and chiral purity by HPLC. The wet 17A wascharged back to the reactor and charged DCM (6 L, 4 vol) to formsuspension. To this 8% NaHCO₃ (6 L, 4 vol) was charged and agitated for30 min at 20±5° C., and let the layers separate. The aqueous phase wasremoved, and the organic layer was washed with brine (3 L, 2 vol). Theorganic phase was concentrated to dryness to yield 17A as a yellow oilthat was dried for at least 72 h under high vacuum and stored at 2-8° C.until used in stage 3. Analysis of crude 17A: Chemical purity: 78.8%(AUC) by HPLC, 96.5% (AUC) by chiral HPLC nm; net weight: 1.54 kg;potency by qNMR: 65.1 wt %; adjusted weight: 1.15 kg (67%); DCM by NMRcontent: 2.22 wt %.

Example 1-12: Preparation of 4-(4-ethynylbenzyl)morpholine hydrochloride(20A)

To a solution of 18A (600 g) in THF (1.2 L) was added a solution ofmorpholine (19A) (0.484 L, 1.2 equiv) in THF (0.60 L) over 79 min at5±5° C. (T: 1.4° C.→7.1° C.). The solution was added at 15±5° C. (T:11.5° C.→19.6° C.) to a slurry of NaBH(OAc)₃ (STAB, 1.173 kg, 1.2 equiv)in THF (4.2 L) over 2 h 43 min. After the addition was complete, thereaction mixture was stirred at 20±5° C. for 14 h and IPC showed 94.7%conversion. Additional morpholine (0.12 equiv) and STAB (0.12 equiv)were charged and batch was stirred for 2 h. IPC showed 100% conversion.8% NaHCO₃ solution (90 mL, 3 vol) was added over 18 min to quench thereaction (T: 20.7° C.→21.8° C.). The mixture was stirred for 49 min. Twophases were separated. The aqueous was extracted with MTBE (2.40 L, 4vol). The combined organics was washed with 5% NaHCO; solution (1.80 L,3 vol), 2 M HCl (2.40 L×1, 1.20 L×1). The combined acidic aqueous wasbasified with 50% NaOH to pH=11.0±0.5 (pH=11.0), extracted with MTBE(2.40 L×2). The organics was washed with basic brine (1.80 L, pH=11.0),dried (sodium sulfate, 0.60 kg), and filtered. Filter cake was washedwith MTBE (0.30 L×2). The combined filtrate was concentrated to 1.80 L.MTBE (2.40 L) was added. The solution was concentrated to 1.80 L andMTBE (2.40 L) was added again. The solution was concentrated to 1.80 L,at this point MTBE/THF (97.4:2.6, v/v). Batch was in-line filtered intothe reactor and MTBE (3.00 L) was used to rinse the container holdingthe batch. To this solution was added 3 M HCl in CPME solution (3.074 L,2.0 equiv) over 1 h 55 min at 5±5° C. The resulting slurry was stirredat 5±5° C. for 2 h (conc. 0.12 mg/mL in mother liquor). The slurry wasfiltered, washed with MTBE (1.80 L×2), and dried to give 20A-HCl (1.038kg, 95% yield, 97.9% (AUC) by HPLC) as a light yellow solid.

Example 1-13: Preparation of(S)-1-(3-(5,6-bis(benzyloxy)pyrimidin-4-yl)-2-(4-((4-(morpholinomethyl)phenyl)ethynyl)phenyl)propyl)azetidine-3-carbonitrile(21A)

20A (0.87 kg, 3.66 mol, 1.05 equiv) was charged into a 30-L reactorunder nitrogen atmosphere. To this, a solution of 17A (2.15 kg, 3.49mol, 1.0 equiv) in sec-BuNH₂ (7.53 L, 3.5 vol) was added with stirringat 20±5° C. The container was rinsed with sec-BuNH₂ (1.075 L, 0.5 vol)and charged the rinses to the batch. No significant exotherm wasobserved. The reaction mixture was cooled to 10±5° C. and water (8.6 mL,4 vol) was charged slowly to the reaction mixture over 1 h maintainingthe batch temperature at 20±5° C. Then nitrogen was bubbled through thereaction mixture for at least 30 min. Pd(Ph₃P)₂Cl₂ (49 g, 0.07 mol, 0.02equiv) was added as a slurry in sec-BuNH₂ (0.11 L, 0.05 vol) whilesparging nitrogen through the reaction mixture. The container was rinsedwith sec-BuNH₂ (0.11 L, 0.05 vol) and charged to the batch. The batchwas gradually heated to 45±5° C. over at least 1 h. The reaction mixturewas stirred at 45±5° C. under nitrogen atmosphere for 12 h. After 12 h,at this point the reaction was deemed to be complete by HPLC (>98%). Thereaction mixture was diluted with IPAc (10.75 L, 5 vol) by quickaddition and stirred for 30 min. Then 5% aqueous citric acid solution(10.75 L, 5 vol) was charged over 30 min by maintaining the batchtemperature at 20±5° C. and stirred for at least 30 min before lettingthe layers separate. Aqueous phase (bottom phase) was removed and theorganic phase was washed with 5% citric acid solution (5 vol×2, 21.5 L).The organic layer was then successively washed with 5% ammoniumpyrrolidinedithiocarbamate (2.3 vol×2, 5 L), DI water (5 vol×1, 10.75L), and 13% brine solution (5 vol×1, 10.75 L). The organic phase wasseparated and filtered through a pad of Celite (≈1.5 inches) to removeinsoluble palladium salts. The reactor used was cleaned and inertedunder nitrogen for 30 min, and charged SiliaMetThiol resin (0.65 kg, 30wt %, Silicycle lot #165304) under nitrogen atmosphere. The organicphase containing 21A in IPAc was then charged with stirring and theslurry was stirred for 16 h at 20±5° C. The slurry was filtered througha pad of Celite (≈1.5 inches). SiliaMetThiol resin (0.65 kg, 30 wt %,Silicycle lot #162798) was charged into the reactor followed by thefiltrate for the 2nd Pd remediation of 21A. The slurry was filteredthrough a pad of Celite (≈1.5 inches) and the filtrate was concentratedunder reduced pressure. The residue was further dried under high vacuumuntil the IPAc content of the batch was less than 10 wt % by ¹H NMR (72h) to afford crude 21A (2.26 kg) as a dark brown gummy oil that was usedin stage 4 without further purification. The batch was split into twoportions for effective drying under high vacuum and the analysis of bothportions is given below. 21A Crude weight: 2.26 kg; Crude purity: 73.5%(AUC) by HPLC; 97.5% (AUC) by chiral HPLC; potency by qNMR: 69.4 wt %;Adjusted weight: 1.57 kg (65%); IPAc content: 7 wt %; Pd content: 428ppm.

Example 1-14: Preparation of(S)-1-(3-(5,6-dihydroxypyrimidin-4-yl)-2-(4-((4-(morpholinomethyl)phenyl)ethynyl)phenyl)propyl)azetidine-3-carbonitrile(Compound A)

A solution of 21A (1.560 kg, 2.26 mol, 1 equiv) in anisole (1.560 L, 1vol,) was added to a mixture of pentamethylbenzene (3.352 kg, 22.6 mol,10.0 equiv,) and TFA (9.750 L, 6.25 vol) at 30±5° C. over a period of 2h. The containers holding 21A were rinsed with anisole (0.195 L×2, 0.125vol×2) and the rinses were added to the reaction mixture at 30±5° C.Batch was stirred at 30° C. for 29 h, at which time HPLC analysis showed95.4% conversion. Batch was then stirred at 20±5° C. for 10.5 h, atwhich time HPLC analysis showed 97.1% conversion. With stirring, batchwas added to MTBE (19.5 L, 12.5 vol) over a period of 1.75 h whilekeeping the temperature below 15° C. The resulting suspension wasstirred at 20±5° C. for 3 h and the supernatant was removed using apump. The precipitate was rinsed with MTBE (4.7 L×2, 3 vol×2) and therinses were removed using a pump. The precipitate was dissolved inacetonitrile (4.68 L, 3 vol) and the pH was adjusted to pH 7.28(measured by a pH meter) using 10.1 L of 8% sodium bicarbonate solutionand 6.0 L of 2 M sodium hydroxide solution. The resulting suspension wasstirred at 20±5° C. for 24.5 h and filtered. The reactor was rinsed withdeionized water (4.68 L×2, 3 vol×2) and the rinses were transferred tothe filter funnel. The filter cake was conditioned under nitrogen for5.5 h to provide a brownish yellow solid (2.762 kg, 26.12 wt % byquantitative NMR). The filter cake was dissolved in acetic acid (2.88 L,4 vol, at 45±5° C. THF (25.0 L, 34.6 vol) was charged at 45±5° C. over aperiod of 5 h 11 min. The resulting suspension was cooled to 20±5° C.over a period of 3 h, stirred at 20±5° C. for 18 h, and filtered. Thereactor was rinsed with 12:1 THF/water (1.44 L, 2 vol) and the rinse wastransferred to the filter funnel. The filter cake was washed with THF(0.72 L×2, 2 vol×2) and dried under vacuum at 45±5° C. for 39.5 h, atwhich time it reached constant weight (0.758 kg). This material wassuspended in water (11.4 L, 15 vol) and treated with 1 M HCl (3.415 L)at 45° C. until a solution was formed at pH˜ 2. Then 1 M NaOH solution(3.40 L) was charged slowly to endpoint of pH 7, at which time batchbecame a slurry. After cooling to 20 t 5° C., the batch was filtered.The reactor was rinsed with water (2.3 L×2, 3 vol×2) and the rinses weretransferred to the filter funnel. The filter cake was dried under vacuumat 45±5° C. for 75 h to provide Compound A [0.745 kg, 65%, 97.5% AUC] asan off-white to light beige solid.

Example 2: Alternative Preparation of Compound 8A

In some instances, Compound 8A is synthesized as outlined in Scheme E.

Example 2-1: Preparation of 4,6-dichloropyrimidin-5-ol (23A)

To a stirred cooled (0° C.) solution of 4,6-dichloro-5-methoxypyrimidine(50.0 g, 0.279 mol) in dichloroethane (500 mL), AlCl₃ (37.2 g, 1.0 eq)was added portionwise over a period of 20 minutes. The reaction mixturewas heated to 50° C. and stirred at that temperature for 2 hr. LCMSindicated the disappearance of starting material. The reaction mixturewas cooled to 0° C. and then quenched with ice cold water (200 mL). Thecompound was extracted with dichloromethane. The aqueous layer wasfurther basified using NaHCO₃ and further extracted withdichloromethane. The organic layers were combined and washed with satd.NaCl and further dried (Na₂SO₄) and concentrated. The crude mixture wasfurther purified on a silica gel column using hexanes in ethyl acetateto afford the product (23.0 g, 50%).

Example 2-2: Preparation of 5-(benzyloxy)-4,6-dichloropyrimidine (24A)

To a stirred solution of 4,6-dichloropyrimidin-5-ol (23A, 22 g, 0.133mol) in acetone (150 mL), K₂CO₃ (46.07 g, 2.5 eq, 0.33 mol) was addedfollowed by the addition of TBAI (5 mol %). The reaction mixture wasstirred at room temperature for a period of 20 minutes, followed by theaddition of benzyl bromide (17.4 mL, 0.146 mol, 1.1 eq) and continuedstirring over a period of 2 hr. LCMS indicated complete conversion. Thereaction mixture was concentrated and then quenched over water (100 mL).The compound was extracted with ethyl acetate (2×125 mL). The organiclayers were combined and washed with saturated NaCl, dried (Na₂SO₄) andconcentrated to dryness. The crude obtained is around 95% pure andyielded (34 g, 100%). No further purification was necessary, and it wastaken to the step.

Example 2-3: Preparation of 4,5-bis(benzyloxy)-6-chloropyrimidine (25A)

To a solution of dried THF (25 mL), NaH (6.4 g, 1.2 eq) was added andcooled to 0° C. using an ice bath. To this BnOH (15.2 mL, 0.146 mol, 1.1eq) dissolved in THF (20 mL) was added dropwise over a period of 30minutes. The reaction mixture was continues stirring for 15 minutes. Tothis, 5-(benzyloxy)-4,6-dichloropyrimidine (24A, 34 g, 0.133 mol)dissolved in THF (50 mL) was added dropwise over a period of 10 minutes.The reaction mixture was stirred at room temperature for a period of 90minutes. LCMS indicated completed conversion. The reaction mixture wasquenched with satd. NH₄Cl and the compound was extracted with ethylacetate (2×125 mL). The organic layers were combined and washed withsatd. NaCl, dried (Na₂SO₄), concentrated to obtain a crude compound. Thecompound was purified using silica gel using hexanes in ethyl acetate toobtain Compound 25A as a colorless oil (34.0 g, 78% yield).

Example 2-4: Preparation of 4,5-bis(benzyloxy)-6-methylpyrimidine (26A)

To a stirred solution of 4,5-bis(benzyloxy)-6-chloropyrimidine (25A, 32g, 0.097 mol) in dioxane (100 mL), potassium acetate (14.4 g, 0.146 mol,1.5 eq), methyl boronic acid (7.63 g, 0.127 mol, 1.3 eq) was added alongwith PdCl₂(PPh₃)₂ (0.05 eq) and reaction mixture was heated to 100° C.over a period of 12 h. LCMS indicated completed conversion. The reactionmixture was diluted with water (75 mL) and extracted with EtOAc (2×75mL). The organic layers were combined and washed with satd. NaCl, dried(Na₂SO₄) and concentrated to give the crude compound. The compound waspurified using silica gel column to obtain Compound 26A a thick oil(24.8 g, 83%).

Example 2-5: Preparation of 4,5-bis(benzyloxy)-6-methylpyrimidine1-oxide (27A)

To m-CPBA (77%, 4.2 g, 18.6 mmol) was added slowly to a stirred solutionof 4,5-bis(benzyloxy)-6-chloropyrimidine (95%, 2.0 g, 6.2 mmol) in DCM(25 mL) at room temperature. The mixture was stirred at room temperaturefor 1 h then treated with solid K₂CO₃ (3.9 g, 6 eq). The resultingmixture was stirred for 10 min then the insolubles removed byfiltration. The filter cake was washed with DCM (3×25 mL) and thefiltrate concentrated in vacuo. Column chromatography (SiO₂ Biotageisolera, 10 g) eluting with 0 to 10% methanol in DCM afforded Compound27A (2.2 g, 95%) as a clear oil which solidified on standing. HPLC-MS(ESI) [M+H]⁺ m/z=323.25; ¹H NMR (500 MHz, Chloroform-d) δ 8.60 (s, 1H),7.53-7.27 (m, 10H), 5.50 (s, 2H), 5.12 (s, 2H), 2.36 (s, 3H).

Example 2-6: Preparation of (5,6-bis(benzyloxy)pyrimidin-4-yl)methanol(8A)

To trifluoroacetic anhydride (0.7 ml, 5.2 mmol) was added to a stirredsolution of 4,5-bis(benzyloxy)-6-methylpyrimidine 1-oxide (500 mg, 1.3mmol) in DCM (8 mL) at room temperature. The mixture was stirredovernight at room temperature then mixed with 2 M aqueous K₂CO₃ solution(20 mL). The resulting biphasic mixture was stirred for 5 min thenextracted with EtOAc (2×25 mL). The combined organic phase was dried(Na₂SO₄) and concentrated in vacuo to leave a crude residue. Columnchromatography (SiO₂ Biotage isolera, 10 g) eluting with 0 to 100% EtOAcin heptanes afforded Compound 8A (330 mg, 79%) as a clear glass whichsolidified on standing. HPLC-MS (ESI) [M+H]⁺ m/z=322.95; ¹H NMR (500MHz, Chloroform-d) δ 8.50 (s, 1H), 7.52-7.46 (m, 2H), 7.43-7.34 (m, 3H),7.34-7.27 (m, 5H), 5.56 (s, 2H), 5.09 (s, 2H), 4.58 (s, 2H).

II. Biological Evaluation Example 3: Bacterial Susceptibility Testing

Minimal inhibitory concentrations (MIC) against a variety ofgram-negative and gram-positive bacterial strains were determined by thebroth microdilution method in accordance with the Clinical andLaboratory Standards Institute (CLSI) guidelines. In brief, organismsuspensions were adjusted to a 0.5 McFarland standard to yield a finalinoculum between 3×10⁵ and 7×10⁵ colony-forming units (CFU)/mL. Drugdilutions and inocula were made in sterile, cation adjustedMueller-Hinton Broth (Beckton Dickinson). An inoculum volume of 100 μLwas added to wells containing 100 μL of broth with 2-fold serialdilutions of drug. All inoculated microdilution trays were incubated inambient air at 35° C. for 18-24 h. Following incubation, the lowestconcentration of the drug that prevented visible growth (OD600 nm<0.05)was recorded as the MIC. Performance of the assay was monitored by theuse of laboratory quality-control strains and levofloxacin, a compoundwith a defined MIC spectrum, in accordance with CLSI guidelines.

Exemplary in vitro assay data against select bacteria for Compound A,Meropenem, and Levofloxacin is provided in Table 1.

TABLE 1 Compound MIC (μg/mL) Bacterium Strain A Meropenem LevofloxacinE. coli ATCC 25922 0.5 0.03 0.03 K. pneumoniae ATCC 13883 0.5 S. aureusATCC 29213 >64 0.125 0.25 E. faecalis ATCC 29212 >64 >1 1 S. pyogenesATCC 12384 >64 0.008 0.5 B. thuringiensis ATCC 35646 >64 0.06 0.125 L.rhamnosus ATCC 53103 >64 >1 1 S. epidermidis ATCC 35984 >64 >1 0.125 B.breve HM 412 >64 >1 4 C. difficile ATCC 700057 >64 1 4 C. sordellii ATCC9714 >64 0.015 1 P. anaerobius DSM 20357 >64 >1 0.5 S. pneumoniae ATCC49619 >64 0.06 1 C. jeikeium NCTC 11914 >64 >1 1 P. acnes ATCC 6919 >640.06 0.5 L. monocytogenes ATCC 7644 >64 0.125 1 N. cyriacigeorgicacomplex NBQAS 3295 >64 >1 8

Compound A has high selectivity for gram-negative bacteria overgram-positive bacteria. Standard of care antibiotics Meropenem andLevofloxacin, in contrast, do have activity against various strains ofgram-positive bacteria.

III. Pharmaceutical Compositions Example 4: Intravenous (I.V.) SolutionFormulation

Compound A is formulated as a solution at a target concentration of 20mg/g of Compound A. The formulation comprises Compound A, SBEβCD(Captisol, sulfobutylether-p-cyclodextrin) or HPβCD(2-hydroxypropyl-β-cyclodextrin), hydrochloric acid (as needed), sodiumhydroxide (as needed), and water. Compound A is added to an aqueoussolution of SBEβCD or HPβCD, and the pH adjusted to 4.2±0.1 usinghydrochloric acid/or sodium hydroxide. The Compound A solution is thenfiltered through a 0.2 μm membrane filter to yield the final solutionformulation.

Table 2 describes the composition of Compound A intravenous solutionformulations, at about 20 mg/g.

TABLE 2 Compound A Osmolarity Formulation Excipient concentration[mg/g]^(a) pH [mOsm/Kg] 3A 2.5% SBEβCD 16.98 4.21 584 3B   5% SBEβCD18.16 4.25 796 3C  10% SBEβCD 19.34 4.26 854 3D 2.5% HPβCD 19.03 4.29527 3E   5% HPβCD 17.62 4.27 599 3F  10% HPβCD 19.06 4.24 659 ^(a)asdetermined by HPLC.

Turbid solutions are observed for all formulations.

The solution formulations can be stored at ambient temperature for up to1 week with no visible changes.

Example 5: Oral Solution Formulations

Compound A is formulated as a solution at a target concentration of ca.40-45 mg/g of Compound A. A stock solutions of ca. 75 mg/g Compound A ina 2:1 mixture of PEG400:propylene glycol, pH 0.5 was diluted intoaqueous vehicles to arrive at the final solution formulations. Afteraddition of the stock solution, the pH of the formulation was adjustedto ca. 3.0-4.0 using NaOH. No precipitation was observed upon pHadjustment.

Table 5 describes the composition of Compound A solution formulations,at about 40-45 mg/g.

TABLE 5 Formulation Formulation Composition pH 6A 40% PEG400, 20% PG, 5%Vitamin E TPGS 3.36 6B 40% PEG400, 20% PG, 5% Vitamin E TPGS + 0.1% HPMC606 3.21 6C 40% PEG400, 20% PG, 5% Vitamin E TPGS + 0.1% Soluplus 2.986D 40% PEG400, 20% PG, 10% SBEβCD 3.67 6E 40% PEG400, 20% PG, 10%SBEβCD + 0.1% HPMC 606 3.10 6F 40% PEG400, 20% PG, 10% SBEβCD + 0.1%Poloxamer 407 3.63 6G 40% PEG400, 20% PG, 20% SBEβCD 3.96

Additional formulations of Compound A at a target concentration of ca.40-45 mg/g of Compound A were prepared as per Table 6 by dissolvingCompound A directly into the vehicle.

TABLE 6 Formulation Formulation Composition 6H 30% SBEβCD + 0.1% HPMC606 6I 0.1M HCl

The examples and embodiments described herein are for illustrativepurposes only and various modifications or changes suggested to personsskilled in the art are to be included within the spirit and purview ofthis application and scope of the appended claims.

1.-51. (canceled)
 52. A process for the preparation of a compound ofFormula 4:

wherein PG is a suitable protecting group; and R is C₁-C₁₀ alkyl;comprising: (1) contacting the compound of Formula 3:

wherein PG is a suitable protecting group; and R is C₁-C₁₀ alkyl; withformamidine acetate and a suitable base in a suitable solvent to providethe compound of Formula
 4. 53. The process of claim 52, wherein: thesuitable base of step (1) is sodium hydride, sodium methoxide, sodiumethoxide, lithium methoxide, lithium ethoxide, n-butyl lithium, lithiumdiisopropylamide (LDA), lithium bis(trimethylsilyl)amide (LiHMDS), orlithium tetramethylpiperidide (LiTMP); and the suitable solvent of step(1b) is methanol, ethanol, isopropyl alcohol, diethyl ether, diisopropylether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, tert-butylmethyl ether, cyclopropyl methyl ether, or a combination thereof. 54.The process of claim 52, wherein: the suitable base of step (1) issodium ethoxide; the suitable solvent of step (1) is ethanol; and step(1) is performed at a temperature of about 0° C. to 10° C. 55.(canceled)
 56. The process of claim 52, further comprising: (2)contacting the compound of Formula 4 with a halogenating agent and asuitable base in a suitable solvent to provide the compound of Formula5:

wherein PG is a suitable protecting group; and R is C₁-C₁₀ alkyl; and X′is Cl or Br.
 57. The process of claim 56, wherein: the halogenatingagent of step (2) is POCl₃, POBr₃, or SOCl₂; the suitable base of step(2) is triethylamine, diisopropylethylamine, sec-butylamine,1,2,2,6,6-pentamethylpiperidine, tributylamine, or1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); and the suitable solvent ofstep (2) is acetonitrile, dichloromethane, chloroform, dichloroethane,toluene, or a combination thereof.
 58. The process of claim 56, wherein:the suitable halogenating agent of step (2) is POCl₃; the suitable baseof step (2) is triethylamine; and the suitable solvent of step (2) istoluene; and step (2) is performed at a temperature of about 85° C. to95° C.
 59. (canceled)
 60. The process of claim 56, further comprising:(3) contacting the compound of Formula 5 with benzyl alcohol in thepresence of a suitable base, and in a suitable solvent to provide acompound of Formula 6-I or Formula 7-I, or a combination thereof:

wherein PG is a suitable protecting group; and R is C₁-C₁₀ alkyl. 61.The process of claim 60, wherein: the suitable base of step (3) istriethylamine, diisopropylethylamine, sec-butylamine,1,2,2,6,6-pentamethylpiperidine, tributylamine, or1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); and the suitable solvent ofstep (3) is acetonitrile, dimethylformamide, diethyl ether, ethanol,tetrahydrofuran, isopropyl alcohol, 1,4-dioxane, toluene, water, or acombination thereof.
 62. The process of claim 60, wherein: the suitablebase of step (3) is DBU; the suitable solvent of step (3) isacetonitrile; and step (3) is performed at a temperature of about 20° C.to 25° C.
 63. (canceled)
 64. The process of claim 60, furthercomprising: (4) contacting the compound of Formula 6-I or Formula 7-I,or combination thereof, with a suitable reducing agent in a suitablesolvent to provide a compound of Formula 8-I:

wherein PG is a suitable protecting group.
 65. The process of claim 64,wherein: the reducing agent of step (4) is sodium borohydride, sodiumcyanoborohydride, sodium triacetoxyborohydride, or lithiumcyanoborohydride; and the suitable solvent of step (4) is acetonitrile,dimethylformamide, diethyl ether, methanol, ethanol, tetrahydrofuran,isopropyl alcohol, 1,4-dioxane, toluene, water, or a combinationthereof.
 66. The process of claim 64, wherein: the reducing agent ofstep (4) is sodium borohydride; and the suitable solvent of step (4) isa mixture of isopropyl alcohol and methanol.
 67. (canceled)
 68. Theprocess of claim 64, further comprising: (5) contacting the compound ofFormula 8-I with a suitable reagent in a suitable solvent to provide thecompound of Formula 9-I:

wherein PG is a suitable protecting group; and LG is a suitable leavinggroup.
 69. The process of claim 68, wherein: the suitable reagent ofstep (5) is a halogenating agent, a sulfonating agent, or a sulfonylchloride; and LG is a halogen.
 70. (canceled)
 71. The process of claim68, wherein: the suitable reagent of step (5) is SOCl₂, PBr₃, or PCl₃;and the suitable solvent is acetonitrile, dimethylformamide, diethylether, ethanol, tetrahydrofuran, isopropyl alcohol, 1,4-dioxane,toluene, water, or a combination thereof.
 72. (canceled)
 73. The processof claim 68, wherein: step (5) further comprises a suitable base,selected from pyridine, N-methylmorpholine, triethylamine,diisopropylethylamine, sec-butylamine, 1,2,2,6,6-pentamethylpiperidine,tributylamine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). 74.(canceled)
 75. The process of claim 52, wherein: R is methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, pentyl,iso-amyl, hexyl, heptyl, octyl, or nonyl.
 76. (canceled)
 77. (canceled)78. The process of claim 52, wherein: PG is benzyl, p-methoxybenzyl,methoxymethyl, [2-(trimethylsilyl)ethoxy]methyl, triisopropylsilyl, ortert-butyldimethylsilyl.
 79. (canceled)
 80. The process of claim 56,wherein: R is ethyl; PG is benzyl; and X′ is Cl. 81.-109. (canceled)110. A compound selected from:

or a salt thereof.